Diagnostic assays for detection of cryptosporidium parvum

ABSTRACT

This invention provides a novel  Cryptosporidium parvum  protein disulfide isomerase polypeptide, and nucleic acids that encode this polypeptide. The inventionalso provides methods, reagents, and kits that are useful for diagnosing infection by  Cryptosporidium parvum . The methods are based on the discovery of binding agents, including recombinant polyclonal antibodies, that bind to the protein disulfide isomerase polypeptide of  C. parvum.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0001] This invention was made with government support under Grant No. 4R44 AI40801-02, awarded by the National Institute of Allergy andInfectious Diseases. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention pertains to the field of diagnostic assays fordetecting infection of an animal by the protozoan parasiteCryptosporidium, in particular, C. parvum. Also provided are novel C.parvum protein disulfide isomerase (PDI) polypeptides, and nucleic acidsencoding the polypeptides.

[0004] 2. Background

[0005] The Cryptosporidium parasites cause infection of a wide varietyof animals, including birds, reptiles, and mammals. C. parvum is theprincipal pathogenic Cryptosporidium species in humans and domesticanimals. C. parvum can cause acute diarrhea in hosts, although inimmunocompetent hosts the disease is self-limiting (Wolfson et al.(1985) N. Engl. J. Med. 312: 1278-1282). In immunocompromised hosts(e.g., AIDS patients), C. parvum can cause a severe and potential lethaldisease (Current et al. (1983) N. Engl. J. Med. 1252-1257; Pitlik et al.(1983) Arch. Intern. Med. 143: 2269-2275; Soave et al. (1984) Ann.Intern. Med. 100: 504-511).

[0006] Cryptosporidium infection typically results from ingestion ofoocysts, which become excystated and release sporozoites. Thesporozoites then infect gut epithelial cells. Once in the epithelialcells, the sporozoites mature into merozoites, which are released andinfect additional epithelial cells. Cryptosporidium also has a sexualcycle, which also occurs in the gut epithelial cells and involves theproduction of sporulated oocysts. Some of the oocysts can becomeexcysted before being shed from the cell. Both sporozoites andmerozoites are found free in the gut.

[0007] Several Cryptosporidium sporozoite and merozoite surface antigenpolypeptides have been reported. For example, five C. parvum surfaceantigens, genes encoding the antigens, and the production of antibodiesagainst C. parvum are discussed in Peterson et al. (1992) Infect. Immun.60: 2343-2348 and PCT patent application PCT/US93/05460 (InternationalPublication No. WO 93/24649). Additional Cryptosporidium surfaceantigens, and production of anti-Cryptosporidium antibodies arediscussed in, for example, Arrowood et al. (1989) Infect. Immun. 57:2283-2288; Bjorneby et al. (1991) Infect. Immun. 59: 1172-1176; Bjornebyet al. (1990) J. Immunol. 145: 298-304; Lumb et al. (1989) Immunol. CellBiol. 67: 267-270; Mead et al. (1988) J. Parasitol. 74: 135-143;Perryman et al. (1990) Infect. Immun. 58: 257-259; Riggs et al. (1989)J. Immunol. 143: 143: 1340-1345; Tilley et al. (1991) Infect. Immun. 59:1002-1007; and Tilley et al. (1990) Can. J. Zool. 68: 1513-1519.

[0008] Diagnosis of Cryptosporidium infection has traditionally involvedmicroscopic detection of ova and parasites (O&P) in stools, which is alaborious process. Other assays have used stains or fluorescent-labeledantibodies which are contacted with a sample, which is then examinedunder a microscope. Both of these assays, however, require subjectiveinterpretation of results. More recently, antigen capture enzymeimmunoassays have been described (e.g., Cypress Diagnostics“Cryptosporidium Ag”; Alexon ProSpecT Cryptosporidium Microplate Assay).

[0009] Therefore, a need exists for improved methods for detectingCryptosporidium infection in animals, including humans. The presentinvention fulfills this and other needs.

SUMMARY OF THE INVENTION

[0010] The present invention provides a novel protein disulfideisomerase (PDI) polypeptide from Cryptosporidium parvum, and nucleicacids that encode the novel PDI. The PDI polypeptides of the inventioninclude an amino acid sequence of at least ten consecutive amino acidsthat are at least substantially identical to a subsequence of an aminoacid sequence as set forth in SEQ ID NO: 3.

[0011] The isolated PDI nucleic acids of the invention include apolynucleotide sequence that encodes an amino acid sequence of which atleast ten consecutive amino acids that are at least substantiallyidentical to a subsequence of an amino acid sequence as set forth in SEQID NO: 3.

[0012] In another embodiment, the invention provides methods ofdiagnosing infection of a mammal by a Cryptosporidium species, inparticular C. parvum. The methods involve contacting a stool sampleobtained from the mammal with a capture reagent that binds to a proteindisulfide isomerase (PDI) of C. parvum. The capture reagent forms acomplex with the PDI if PDI is present in the test sample. The presenceor absence of the PDI bound to the capture reagent is then detected; thepresence of the PDI is indicative of Cryptosporidium infection of themammal.

[0013] The invention also provides devices and kits for diagnosinginfection of a mammal by a Cryptosporidium species, in particular C.parvum. The kits typically include, inter alia, a solid support uponwhich is immobilized a capture reagent which binds to a PDI of C.parvum, and a detection reagent which binds to the PDI.

[0014] Also provided by the invention are recombinant monoclonal andpolyclonal antibodies that bind to C. parvum PDI.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIGS. 1A-C show a top piece of an apparatus for performing animmunoassay for detecting C. parvum infection in a sample. FIG. 1A is atop view, showing an elongated well in the center. FIG. 1B is a sectionview of the top piece, showing a membrane that is ultrasonically weldedto the underside of the top piece. FIG. 1C is an end view of the toppiece of the apparatus.

[0016] FIGS. 2A-C show a bottom piece of an apparatus for performing animmunoassay for detecting C. parvum infection in a sample. FIG. 2A is atop view, FIG. 2B is a section view, and FIG. 2C is an end view of thebottom piece. To construct a complete apparatus, a bottom piece isjoined to a top piece such as is shown in FIGS. 1A-C.

[0017]FIG. 3A presents the nucleotide sequence of a Cryptosporidiumparvum protein disulfide isomerase (PDI) cDNA (SEQ ID NO: 1). Thesequence differs from a PDI nucleotide sequence reported by Blunt et al.((1996) Gene 181: 221-223) in two locations. First, the sequencedescribed herein includes a guanine residue at position 766 (bold) thatis not present in the Blunt et al. sequence. Second, the sequencedescribed herein is lacking a cytosine after position 860 (shown as abolded underline). FIG. 3B shows the deduced amino acid sequence of theC. parvum PDI (SEQ ID NO: 2). A 32 amino acid region of the amino acidsequence (shown in bold) differs from the amino acid sequence for PDIreported by Blunt et al., supra.

DETAILED DESCRIPTION

[0018] Definitions

[0019] The phrases “specifically binds to” or “specificallyimmunoreactive with”, when referring to an antibody or other bindingmoiety refers to a binding reaction which is determinative of thepresence of a target antigen in the presence of a heterogeneouspopulation of proteins and other biologics. Thus, under designated assayconditions, the specified binding moieties bind preferentially to aparticular target antigen and do not bind in a significant amount toother components present in a test sample. Specific binding to a targetantigen under such conditions may require a binding moiety that isselected for its specificity for a particular target antigen. A varietyof immunoassay formats may be used to select antibodies that arespecifically immunoreactive with a particular protein. For example,solid-phase ELISA immunoassays are routinely used to select monoclonalantibodies specifically immunoreactive with an antigen. See Harlow andLane (1988) Antibodies, A Laboratory Manual, Cold Spring HarborPublications, New York, for a description of immunoassay formats andconditions that can be used to determine specific immunoreactivity.Typically a specific or selective reaction will be at least twicebackground signal or noise and more typically more than 10 to 100 timesbackground. Specific binding between an antibody or other binding agentand an antigen means a binding affinity of at least 10⁶ M⁻¹. Preferredbinding agents bind with affinities of at least about 10⁷ M⁻¹, andpreferably 10⁸ M⁻¹ to 10⁹ M⁻¹ or 10¹⁰ M⁻¹.

[0020] The term “epitope” means an antigenic determinant capable ofspecific binding to an antibody. Epitopes usually consist of chemicallyactive surface groupings of molecules such as amino acids or sugar sidechains and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics.Conformational and nonconformational epitopes are distinguished in thatthe binding to the former but not the latter is lost in the presence ofdenaturing solvents.

[0021] The basic antibody structural unit is known to comprise atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 Kda). The amino-terminal portion of eachchain includes a variable region of about 100 to 110 or more amino acidsprimarily responsible for antigen recognition. The carboxy-terminalportion of each chain defines a constant region primarily responsiblefor effector function.

[0022] Light chains are classified as either kappa or lambda. Heavychains are classified as gamma, mu, alpha, delta, or epsilon, and definethe antibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively.Within light and heavy chains, the variable and constant regions arejoined by a “J” region of about 12 or more amino acids, with the heavychain also including a “D” region of about 10 more amino acids. (Seegenerally, Fundamental Immunology (See, e.g., Paul, FundamentalImmunology, 3^(rd) Ed., 1993, Raven Press, New York).

[0023] The variable regions of each light/heavy chain pair form theantibody binding site. The chains all exhibit the same general structureof relatively conserved framework regions (FR) joined by threehypervariable regions, also called complementarily determining regionsor CDRs. The CDRs from the two chains of each pair are aligned by theframework regions, enabling binding to a specific epitope. CDR and FRresidues are delineated according to the standard sequence definition ofKabat et al., supra. An alternative structural definition has beenproposed by Chothia et al. (1987) J. Mol. Biol. 196: 901-917; (1989)Nature 342: 878-883; and (1989) J. Mol. Biol. 186: 651-663.

[0024] The term “antibody” is used to mean whole antibodies and bindingfragments thereof. Binding fragments include single chain fragments, Fvfragments and Fab fragments The term Fab fragment is sometimes used inthe art to mean the binding fragment resulting from papain cleavage ofan intact antibody. The terms Fab′ and F(ab′ )₂ are sometimes used inthe art to refer to binding fragments of intact antibodies generated bypepsin cleavage. Here, “Fab” is used to refer generically to doublechain binding fragments of intact antibodies having at leastsubstantially complete light and heavy chain variable domains sufficientfor antigen-specific bindings, and parts of the light and heavy chainconstant regions sufficient to maintain association of the light andheavy chains. Usually, Fab fragments are formed by complexing afull-length or substantially full-length light chain with a heavy chaincomprising the variable domain and at least the CH1 domain of theconstant region.

[0025] An isolated species or population of species means an objectspecies (e.g., binding polypeptides of the invention) that is thepredominant species present (i.e., on a molar basis it is more abundantthan other species in the composition). Preferably, an isolated speciescomprises at least about 50, 80 or 90 percent (on a molar basis) of allmacromolecular species present. Most preferably, the object species ispurified to essential homogeneity (contaminant species cannot bedetected in the composition by conventional detection methods).

[0026] The terms “identical” or percent “identity,” in the context oftwo or more nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the following sequence comparison algorithms or by visual inspection.

[0027] The phrase “substantially identical,” in the context of twonucleic acids, refers to two or more sequences or subsequences that haveat least 80%, preferably 85%, most preferably 90-95% nucleotideidentity, when compared and aligned for maximum correspondence, asmeasured using one of the following sequence comparison algorithms or byvisual inspection. For amino acid sequences, “substantial identical”refers to two or more sequences or subsequences that have at least 60%identity, preferably 75% identity, and more preferably 90-95% identify,when compared and aligned for maximum correspondence, as measured usingone of the following sequence comparison algorithms or by visualinspection. Preferably, the substantial identity exists over a region ofthe nucleic acid or amino acid sequences that is at least about 10residues in length, more preferably over a region of at least about 20residues, and most preferably the sequences are substantially identicalover at least about 100 residues. In a most preferred embodiment, thesequences are substantially identical over the entire length of thespecified regions (e.g., coding regions).

[0028] For sequence comparison, typically one sequence acts as areference sequence, to which test sequences are compared. When using asequence comparison algorithm, test and reference sequences are inputinto a computer, subsequence coordinates are designated, if necessary,and sequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

[0029] Optimal alignment of sequences for comparison can be conducted,e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl.Math. 2:482 (1981), by the homology alignment algorithm of Needleman &Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity methodof Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by visual inspection (seegenerally, Current Protocols in Molecular Biology, F. M. Ausubel et al.,eds., Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc., (1995 Supplement)(Ausubel)).

[0030] Examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1990) J. Mol. Biol.215: 403-410 and Altschuel et al. (1977) Nucleic Acids Res. 25:3389-3402, respectively. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation (http://www.ncbi.nlm.nih.gov/). This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold (Altschul et al, supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are then extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlength(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0031] In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

[0032] A further indication that two nucleic acid sequences orpolypeptides are substantially identical is that the polypeptide encodedby the first nucleic acid is immunologically cross reactive with thepolypeptide encoded by the second nucleic acid, as described below.Thus, a polypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions. Another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions, as described below.

[0033] “Conservatively modified variations” of a particularpolynucleotide sequence refers to those polynucleotides that encodeidentical or essentially identical amino acid sequences, or where thepolynucleotide does not encode an amino acid sequence, to essentiallyidentical sequences. Because of the degeneracy of the genetic code, alarge number of functionally identical nucleic acids encode any givenpolypeptide. For instance, the codons CGU, CGC, CGA, CGG, AGA, and AGGall encode the amino acid arginine. Thus, at every position where anarginine is specified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations,” which are onespecies of “conservatively modified variations.” Every polynucleotidesequence described herein which encodes a polypeptide also describesevery possible silent variation, except where otherwise noted. One ofskill will recognize that each codon in a nucleic acid (except AUG,which is ordinarily the only codon for methionine) can be modified toyield a functionally identical molecule by standard techniques.Accordingly, each “silent variation” of a nucleic acid which encodes apolypeptide is implicit in each described sequence.

[0034] Furthermore, one of skill will recognize that individualsubstitutions, deletions or additions which alter, add or delete asingle amino acid or a small percentage of amino acids (typically lessthan 5%, more typically less than 1%) in an encoded sequence are“conservatively modified variations” where the alterations result in thesubstitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. The following five groups each containamino acids that are conservative substitutions for one another:

[0035] Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L),Isoleucine (I);

[0036] Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

[0037] Hydroxy: Serine (S), Threonine (T);

[0038] Sulfur-containing: Methionine (M), Cysteine (C);

[0039] Basic: Arginine (R), Lysine (K), Histidine (H);

[0040] Acidic: Aspartic acid (D), Glutamic acid (E);

[0041] Amide: Asparagine (N), Glutamine (Q).

[0042] See also, Creighton (1984) Proteins, W.H. Freeman and Company. Inaddition, individual substitutions, deletions or additions which alter,add or delete a single amino acid or a small percentage of amino acidsin an encoded sequence are also “conservatively modified variations”.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] The invention provides novel Cryptosporidium parvum proteindisulfide isomerase (PDI) polypeptides, and nucleic acids that encodethese polypeptides. Also provided by the invention are methods,reagents, and kits that are useful for diagnosing infection of a mammalby a Cryptosporidium species, in particular C. parvum. The assaysprovide a rapid, accurate and cost-effective means for detectingCryptosporidium infection. The methods of the invention are bothsensitive and specific, and can be used for detecting a Cryptosporidiumantigen that is soluble.

[0044] The methods, compositions and kits provided by the instantinvention are useful for detecting Cryptosporidium infection in testsamples, including biological samples such as cultures, tissue samples,bodily fluids, and the like. Typically, the biological sample analyzedfor Cryptosporidium infection will be a stool sample. For liquid orsemi-solid stool samples, a portion of the sample is added to an assaycontainer and, optionally, diluted with a suitable diluent such as wateror an appropriate buffer and mixed. Suitable buffers include, forexample, buffered protein solutions and the like. Solid stool samplescan be placed in a diluent and suspended by vigorous mixing. Typically,the sample is diluted sufficiently to provide a solution of suitableclarity for use in the assays; this is generally about a 3-20 folddilution, with about a 10-fold dilution being typical. After mixing, onecan clarify the sample by, for example, filtration or centrifugation orother methods known to those of skill in the art. In general, well knownmethods for preparing test samples for assays, such as immunoassays, aresuitable for preparing test samples for analysis using the methodsprovided by the invention.

[0045] A. C. parvum Protein Disulfide Isomerase (PDI) Nucleic Acids andPolypeptides

[0046] The invention provides novel isolated PDI polypeptides, andisolated nucleic acids that encode the PDI polypeptides.

[0047] 1. C. parvum Protein Disulfide Isomerase Polypeptides The presentinvention provides novel C. parvum protein disulfide isomerase (PDI)polypeptides. The polypeptides are useful for several purposes. Forexample, one can use the PDI polypeptides of the invention to facilitatethe folding of disulfide-containing proteins, e.g., proteins producedusing recombinant methods. The polypeptides are also useful asimmunogens for producing antibodies against PDI; such antibodies finduse in immunoassays, for purification of PDI, and other uses.

[0048] The PDI polypeptides of the invention have many uses, includinguse as immunogens for producing antibodies against PDI. The nucleicacids of the invention find use for recombinant expression of PDI, foridentifying protein disulfide isomerases from other species, and forother purposes known to those of skill in the art.

[0049] The amino acid sequence of a C. parvum PDI polypeptide of theinvention is shown in FIG. 3. The amino acid sequence of the PDIpolypeptides of the invention differ substantially from a C. parvum PDIpredicted amino acid sequence that had been reported previously (Bluntet al. (1996) Gene 181: 221-223; Genbank Accession No. U48261).Specifically, a 32 amino acid region of the PDI sequence of the presentinvention is completely different from that of Blunt et al., as shownbelow. The 32 amino acid region that differs between the two sequencesis highlighted in bold type, and the numbering at the beginning andending of the sequence corresponds to the numbering convention used byBlunt et al. Applicants 250 SREGYTAWFCGTNEDFAKYASNIRKVAADYREKYAFVFLDT290 (SEQ ID NO:3) Blunt et al. 250SREGYTPGSVVLTRTSPSMLQTLERLQLITEKSMPLFSLDT 290 (SEQ ID NO:4)

[0050] Accordingly, the present invention provides isolated proteindisulfide isomerase polypeptides that include an amino acid sequence ofwhich at least ten consecutive amino acids are at least substantiallyidentical to a subsequence of the amino acid sequenceAWFCGTNEDFAKYASNIRKVAADYR EKYAFVF (SEQ ID NO: 3). More preferably, thePDI polypeptides of the invention include at least 15, more preferablyat least 20, still more preferably at least about 25-32 amino acids thatare substantially identical to the amino acid sequence set forth in SEQID NO: 3. In a particularly preferred embodiment, the PDI polypeptidesof the invention include an amino acid sequence that is identical to theamino acid sequence of SEQ ID NO: 3, or a subsequence thereof.

[0051] Included in the invention are isolated PDI polypeptides that areat least substantially identical to a PDI polypeptide having an aminoacid sequence as set forth in SEQ ID NO: 2, which provides thefill-length PDI polypeptide.

[0052] The PDI polypeptides of the invention can be produced by methodsknown to those of skill in the art. In a preferred embodiment, the PDIproteins or subsequences thereof are synthesized using recombinant DNAmethodology. Generally this involves creating a DNA sequence thatencodes the polypeptide, modified as desired, placing the DNA in anexpression cassette under the control of a particular promoter,expressing the protein in a host, isolating the expressed protein and,if required, renaturing the protein.

[0053] The polypeptides of the invention can be expressed in a varietyof host cells, including E. coli, other bacterial hosts, yeasts,filamentous fungi, and various higher eukaryotic cells such as the COS,CHO and HeLa cells lines and myeloma cell lines. Techniques for geneexpression in microorganisms are described in, for example, Smith, GeneExpression in Recombinant Microorganisms (Bioprocess Technology, Vol.22), Marcel Dekker, 1994. Examples of bacteria that are useful forexpression include, but are not limited to, Escherichia, Enterobacter,Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsielia, Proteus,Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, and Paracoccus.Filamentous fungi that are useful as expression hosts include, forexample, the following genera: Aspergillus, Trichoderma, Neurospora,Penicillium, Cephalosporium, Achlya, Podospora, Mucor, Cochliobolus, andPyricularia. See, e.g., U.S. Pat. No. 5,679,543 and Stahl and Tudzynski,Eds., Molecular Biology in Filamentous Fungi, John Wiley & Sons, 1992.Synthesis of heterologous proteins in yeast is well known and describedin the literature. Methods in Yeast Genetics, Sherman, F., et aL, ColdSpring Harbor Laboratory, (1982) is a well recognized work describingthe various methods available to produce the enzymes in yeast.

[0054] A polynucleotide that encodes a PDI polypeptide of the inventioncan be operably linked to appropriate expression control sequences for aparticular host cell in which the polypeptide is to be expressed. Suchconstructs are often referred to as “expression cassettes.” For E. coli,appropriate control sequences include a promoter such as the T7, trp, orlambda promoters, a ribosome binding site and preferably a transcriptiontermination signal. For eukaryotic cells, the control sequencestypically include a promoter which optionally includes an enhancerderived from immunoglobulin genes, SV40, cytomegalovirus, etc., and apolyadenylation sequence, and may include splice donor and acceptorsequences. In yeast, convenient promoters include GAL 1,10 (Johnson andDavies (1984) Mol. Cell. Biol. 4:1440-1448) ADH2 (Russell et al. (1983)J. Biol. Chem. 258:2674-2682), PHO5 (EMBO J. (1982) 6:675-680), and MFα1(Herskowitz and Oshima (1982) in The Molecular Biology of the YeastSaccharomyces (eds. Strathem, Jones, and Broach) Cold Spring HarborLab., Cold Spring Harbor, N.Y., pp. 181-209).

[0055] Expression cassettes are typically introduced into a vector whichfacilitates entry into a host cell, and maintenance of the expressioncassette in the host cell. Vectors that include a polynucleotide thatencodes a PDI polypeptide are provided by the invention. Such vectorsoften include an expression cassette that can drive expression of thePDI polypeptide. To easily obtain a vector of the invention, one canclone a polynucleotide that encodes the PDI polypeptide into acommercially or commonly available vector. A variety of common vectorssuitable for this purpose are well known in the art. For cloning inbacteria, common vectors include pBR322 derived vectors such aspBLUESCRIPT™, and λ-phage derived vectors. In yeast, vectors includeYeast Integrating plasmids (e.g., YIp5) and Yeast Replicating plasmids(the YRp series plasmids) and pGPD-2. A multicopy plasmid with selectivemarkers such as Leu-2, URA-3, Trp-1, and His-3 is also commonly used. Anumber of yeast expression plasmids such as YEp6, YEpl3, YEp4 can beused as expression vectors. The above-mentioned plasmids have been fullydescribed in the literature (Botstein et al. (1979) Gene 8:17-24; Broachet al. (1979) Gene, 8:121-133). For a discussion of yeast expressionplasmids, see, e.g., Parents, B., YEAST (1985), and Ausubel, Sambrook,and Berger, all supra). Expression in mammalian cells can be achievedusing a variety of commonly available plasmids, including pSV2, pBC12BI,and p91023, as well as lytic virus vectors (e.g., vaccinia virus,adenovirus, and baculovirus), episomal virus vectors (e.g., bovinepapillomavirus), and retroviral vectors (e.g., murine retroviruses).

[0056] The nucleic acids that encode the polypeptides of the inventioncan be transferred into the chosen host cell by well-known methods suchas calcium chloride transformation for E. coli and calcium phosphatetreatment or electroporation for mammalian cells. Cells transformed bythe plasmids can be selected by resistance to antibiotics conferred bygenes contained on the plasmids, such as the amp, gpt, neo and hyggenes, among others. Techniques for transforming fungi are well known inthe literature and have been described, for instance, by Beggs et al.((1978) Proc. Natl. Acad. Sci. USA 75: 1929-1933), Yelton et al. ((1984)Proc. Natl. Acad. Sci. USA 81: 1740-1747), and Russell ((1983) Nature301: 167-169). Procedures for transforming yeast are also well known(see, e.g., Beggs (1978) Nature (London), 275:104-109; and Hinnen et al.(1978) Proc. Natl. Acad. Sci. USA, 75:1929-1933. Transformation andinfection methods for mammalian and other cells are described in Bergerand Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology152 Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al.(1989) Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3, ColdSpring Harbor Laboratory, Cold Spring Harbor Press, N.Y., (Sambrook etal.); Current Protocols in Molecular Biology, F. M. Ausubel et al.,eds., Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc., (1994 Supplement)(Ausubel).

[0057] Once expressed, the PDI proteins can be purified, eitherpartially or substantially to homogeneity, according to standardprocedures of the art, such as, for example, ammonium sulfateprecipitation, affinity columns, column chromatography, gelelectrophoresis and the like (see, generally, R. Scopes, ProteinPurification, Springer-Verlag, N.Y. (1982), Deutscher, Methods inEnzymology Vol. 182:Guide to Protein Purification., Academic Press, Inc.N.Y. (1990)). Once purified, partially or to homogeneity as desired, thepolypeptides may then be used (e.g., in screening assays for modulatorsfor gene expression or as immunogens for antibody production).

[0058] One of skill in the art would recognize that after chemicalsynthesis, biological expression, or purification, the PDI protein(s)may possess a conformation substantially different than the nativeconformations of the constituent polypeptides. In this case, it may benecessary to denature and reduce the polypeptide and then to cause thepolypeptide to re-fold into the preferred conformation. Methods ofreducing and denaturing proteins and inducing re-folding are well knownto those of skill in the art (See, Debinski et al. (1993) J. Biol.Chem., 268: 14065-14070; Kreitman and Pastan (1993) Bioconjug. Chem., 4:581-585; and Buchner, et al., (1992) Anal. Biochem., 205: 263-270).Debinski et al., for example, describe the denaturation and reduction ofinclusion body proteins in guanidine-DTE. The protein is then refoldedin a redox buffer containing oxidized glutathione and L-arginine.

[0059] One of skill also would recognize that modifications can be madeto the PDI polypeptides without diminishing their biological activity.Some modifications may be made to facilitate the cloning, expression, orincorporation of the polypeptide into a fusion protein. Suchmodifications are well known to those of skill in the art and include,for example, a methionine added at the amino terminus to provide aninitiation site, or additional amino acids (e.g., poly His) placed oneither terminus to create conveniently located restriction sites ortermination codons or purification sequences.

[0060] 2. Antibodies that Specifically Bind C. parvum Protein DisulfideIsomerase

[0061] The invention also provides antibodies that can specifically bindC. parvum PDI polypeptides of the invention. These antibodies can beprepared using as immunogens natural, recombinant or syntheticpolypeptides of 10 amino acids in length, or greater, selected fromamino acid subsequences of the amino acid sequences shown in SEQ ID NO:2 and SEQ ID NO: 3. Such polypeptides can function as immunogens(antigens) that can be used for the production of monoclonal orpolyclonal antibodies. In one class of preferred embodiments, animmunogenic peptide conjugate is also included as an immunogen.Naturally occurring polypeptides are also used either in pure or impureform. Production of antibodies against PDI polypeptides of the inventionis discussed in more detail below. In some embodiments, the antibodiesof the invention can bind to polypeptides that include the amino acidsequence as shown in SEQ ID NO: 3, but do not bind substantially to PDIpolypeptides that lack this amino acid sequence.

[0062] 3. Nucleic Acids encoding C. parvum Protein Disulfide Isomerase

[0063] The invention also provides isolated and/or recombinant nucleicacids that encode the PDI polypeptides of the invention, and functionaldomains thereof. The nucleic acids are useful for many purposes. Forexample, one can use the PDI nucleic acids of the invention to producePDI polypeptides. The nucleic acids of the invention are also useful asprobes to identify PDI-encoding nucleic acids in human tissues andsamples, and also in those of other mammals that can become infected byCryptosporidium. The nucleic acids are also useful to study theexpression of PDI, both in vitro and in vivo.

[0064] In one embodiment, the invention provides isolated nucleic acidsthat include a polynucleotide sequence that encodes a polypeptide thathas an amino acid sequence of which at least ten consecutive amino acidsare at least substantially identical to a subsequence of an amino acidsequence as set forth in SEQ ID NO: 3. Included in the invention arenucleic acids that encode a full-length PDI polypeptide that has anamino acid sequence that is substantially identical to the amino acidsequence set forth in SEQ ID NO: 2. In one presently preferredembodiment, the nucleic acids have the polynucleotide sequence as setforth in SEQ ID NO: 1.

[0065] The PDI nucleic acids of the invention can be isolated, forexample, by routine cloning methods. The cDNA sequence provided in SEQID NO: 1 can be used to provide probes that specifically hybridize to aPDI gene, to a PDI MRNA, or to a PDI cDNA in a cDNA library (e.g., in aSouthern or Northern blot). Once the target PDI nucleic acid isidentified, it can be isolated according to standard methods known tothose of skill in the art (see, e.g., Sambrook, Berger, and Ausubel,supra.). In another preferred embodiment, the PDI nucleic acids of theinvention can be isolated by amplification methods such as polymerasechain reaction (PCR), the ligase chain reaction (LCR), thetranscription-based amplification system (TAS), the self-sustainedsequence replication system (S SR). A wide variety of cloning and invitro amplification methodologies are well-known to persons of skill.Examples of these techniques and instructions sufficient to directpersons of skill through many cloning exercises are found in Berger,Sambrook, and Ausubel (all supra.); Cashion et al., U.S. Pat. No.5,017,478; and Carr, European Patent No. 0,246,864. Examples oftechniques sufficient to direct persons of skill through in vitroamplification methods are found in Berger, Sambrook, and Ausubel, aswell as Mullis et al. (1987) U.S. Pat. No. 4,683,202; PCR Protocols AGuide to Methods and Applications (Innis et al., eds) Academic PressInc. San Diego, Calif. (1990) (Innis); Arnheim & Levinson (Oct. 1, 1990)C&EN 36-47; The Journal Of NIH Research (1991) 3: 81-94; (Kwoh et al.(1989) Proc. Nat'l. Acad. Sci. USA 86: 1173; Guatelli et al. (1990)Proc. Natl. Acad. Sci. USA 87: 1874; Lomell etal. (1989) J. Clin. Chem.35: 1826; Landegren et al. (1988) Science 241: 1077-1080; Van Brunt(1990) Biotechnology 8: 291-294; Wu and Wallace (1989) Gene, 4: 560; andBarringer et al. (1990) Gene 89: 117.

[0066] The invention also provides nucleic acid constructs in which aPDI polynucleotide of the invention is operably linked to a promoterthat is functional in a desired host cell. Such constructs are oftenprovided as an “expression cassette”, which can also include othersequences involved in transcription, translation, and post-translationalmodification of the PDI polypeptide. Examples of suitable promoters andother control sequences are described herein. The invention alsoprovides expression vectors, and host cells that comprise the PDInucleic acids of the invention.

[0067] In presently preferred embodiments, the PDI-encoding nucleicacids of the invention have a translation initiation codon (generallyATG, or AUG in the mRNA) that is in frame with codons that encode theamino acid sequence set forth in SEQ ID NO: 3. Thus, a PDI polypeptideexpressed using a PDI nucleic acid of the invention will preferablyinclude the amino acid sequence of SEQ ID NO: 3.

[0068] B. Assay Reagents

[0069] The assays of the invention involve detecting the presence in abiological sample of a Cryptosporidium parvum protein disulfideisomerase (PDI), which is an antigen that is specific forCryptosporidium. The invention provides assay reagents that are capableof specifically binding to the PDI antigen. These assay reagents can beused in one or more steps of the assay. For example, the assay reagentscan be immobilized on a solid support and used to immobilize PDI on asolid support. Assay reagents can also be used to detect theCryptosporidium antigens by, for example, attaching a detectable labelto a binding moiety that binds to PDI. These are discussed in greaterdetail below.

[0070] The assay means for detecting PDI are, in some embodiments,binding assays. In these assays, which include immunoassays, PDI isdetected using detection reagents that are capable of specificallybinding to PDI. The detection reagents include at least a binding moietyand a detectable label. Suitable binding moieties include any moleculethat is capable of specifically binding to PDI. Antibodies and fragmentsthereof are examples of binding components that are suitable for use indetection moieties.

[0071] Various procedures known in the art can be used for theproduction of antibodies that specifically bind to PDI. For theproduction of polyclonal antibodies, one can use PDI to inoculate any ofvarious host animals, including but not limited to rabbits, mice, rats,sheep, goats, and the like. The PDI polypeptide can be prepared byrecombinant means as described above using an expression vectorcontaining a gene encoding the polypeptide; the complete nucleotidesequence is presented in SEQ ID NO: 1.

[0072] Monoclonal antibodies can be prepared by any technique thatprovides for the production of antibody molecules by continuous celllines in culture, including the hybridoma technique originally developedby Kohler and Milstein ((1975) Nature 256: 495-497), as well as thetrioma technique, the human B-cell hybridoma technique (Kozbor et al.(1983) Immunology Today 4: 72), and the EBV-hybridoma technique toproduce human monoclonal antibodies (Cole et al. (1985) in MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).Monoclonal antibodies also can be produced in germ-free animals as wasdescribed in PCT/US89/02545 (Publication No. WO8912690, published Dec.12, 1989) and U.S. Pat. No. 5,091,512.

[0073] Fragments of antibodies are also useful as binding moieties.While various antibody fragments can be obtained by the digestion of anintact antibody, one of skill will appreciate that such fragments may besynthesized de novo either chemically or by utilizing recombinant DNAmethodology. Thus, the term “antibody,” as used herein, also includesantibody fragments either produced by the modification of wholeantibodies or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv). Single chain antibodies are alsouseful to construct detection moieties. Methods for producing singlechain antibodies were described in, for example, U.S. Pat. No.4,946,778. Techniques for the construction of Fab expression librarieswere described by Huse et al. (1989) Science 246: 1275-1281; thesetechniques facilitate rapid identification of monoclonal Fab fragmentswith the desired specificity for PDI. Suitable binding moieties alsoinclude those that are obtained using methods such as phage display.

[0074] To prepare a suitable antigen preparation, one can prepare a cDNAexpression library from C. parvum and screen the library with apolyclonal antibody that is raised against a crude preparation of PDI.The cDNA inserts from those expression plasmids that express the PDI arethen subcloned and sequenced. The PDI-encoding inserts are cloned intoan expression vector and use to transform E. coli or other suitable hostcells. The resulting preparation of recombinant PDI is then used toinoculate an animal, e.g., a mouse.

[0075] In preferred embodiments, the assay reagents use recombinantlyproduced polyclonal or monoclonal antibodies that bind to the PDI asbinding moieties. Recombinant antibodies are typically produced byimmunizing an animal with the PDI, obtaining RNA from the spleen orother antibody-expressing tissue of the animal, making cDNA, amplifyingthe variable domains of the heavy and light immunoglobulin chains,cloning the amplified DNA into a phage display vector, infecting E.coli, expressing the phage display library, and selecting those librarymembers that express an antibody that binds to PDI. Methods suitable forcarrying out each of these steps are described in, for example U.S.patent application Ser. No. 08/835,159, filed Apr. 4, 1997. In preferredembodiments, the antibody or other binding peptides are expressed on thecell surface of a replicable genetic unit, such as a filamentous phage,and especially phage M13, Fd and F1. Most work has inserted librariesencoding polypeptides to be displayed into either gIII or gVIII of thesephage, forming a fusion protein which is displayed on the surface of thephage. See, e.g., Dower, WO 91/19818; Devlin, WO 91/18989; MacCafferty,WO 92/01047 (gene III); Huse, WO 92/06204; Kang, WO 92/18619 (geneVIII).

[0076] In a preferred embodiment, the genes that encode the heavy andlight chains of antibodies present in the cDNA library are amplifiedusing a set of primers that can amplify substantially all of thedifferent heavy and light chains. The resulting amplified fragments thatresult from the amplification step are pooled and subjected toasymmetric PCR so that only one strand (e.g., the antisense strand) isamplified. The single strand products are phosphorylated, annealed to asingle-stranded uracil template (e.g., the vector BS45, described inU.S. patent application Ser. No. 08/835,159, which has coding regionsfor the constant regions of mouse heavy and light chains), andintroduced into a uracil DNA glycosylase⁺ host cell to enrich forvectors that contain the coding sequences for heavy and light chainvariable domains.

[0077] To screen for phage that express an antibody that binds to PDI,one can attach a label to PDI using methods known to those of skill inthe art. In a preferred embodiment, the phage that display suchantibodies are selected using PDI to which is attached an immobilizabletag, e.g., biotin. The phage are contacted with the biotinylatedantigen, after which the phage are selected by contacting the resultingcomplex with avidin attached to a magnetic latex bead or other solidsupport. The selected phage are then plated, and may be screened withPDI to which is attached a detectable label.

[0078] In a preferred embodiment, the library is enriched for thosephage that display more than one antibody that binds to PDI. Methods andvectors that are useful for this enrichment are described in U.S. patentapplication Ser. No. 08/835,159. The panning can be repeated one or moretimes to enhance the specificity and sensitivity of the resultingantibodies. Preferably, panning is continued until the percentage offunctional positives is at least about 70%, more preferably at leastabout 80%, and most preferably at least about 90%.

[0079] A recombinant anti-PDI monoclonal antibody can then be selectedby amplifying antibody-encoding DNA from individual plaques, cloning theamplified DNA into an expression vector, and expressing the antibody ina suitable host cell (e.g., E. coli). The antibodies are then tested forability to bind PDI. An example of a recombinant monoclonal antibodyprepared using this method is the mAb CP.2, which was deposited underthe Budapest Treaty with the American Type Culture Collection (10801University Boulevard, Manassas, Va. 20110-2209) on ______, and has beenassigned ATCC Accession No. ______.

[0080] Recombinant polyclonal antibodies are particularly preferred, inparticular because of the various forms of PDI that may be found inclinical samples due to, for example, proteolysis. The diverse finebinding specificity of members of a population of polyclonal antibodiesoften allows the population to bind to several forms of PDI (e.g.,species variants, escape mutant forms, proteolytic fragments) to which amonoclonal reagent may be unable to bind. Methods for producingrecombinant polyclonal antibodies are described in co-pending, commonlyassigned U.S. patent application Ser. No. 08/835,159, filed Apr. 4,1997. Specific methods of producing recombinant polyclonal antibodiesthat bind to PDI are described in the Examples below.

[0081] Polyclonal antibodies can be prepared as described above, exceptthat an individual antibody is not selected. Rather, the pool of phageare used for the screening, preferably using an equal number of phagefrom each sample. In preferred embodiments, the phage are enriched forthose that display more than one copy of the respective antibodies. Thephage are then selected for those that bind to PDI. For example, one canuse a biotinylated anti-PDI monoclonal antibody and PDI to concentratethose phage that express antibodies that bind to PDI. The biotinylatedmonoclonal antibody is immobilized on a solid support (e.g., magneticlatex) to which is attached avidin. The phage that are bound to theimmobilized PDI are eluted, plated, and the panning repeated until thedesired percentage of functional positives is obtained.

[0082] C. Assay Formats

[0083] The assays for detecting Cryptosporidium infection can beperformed in any of several formats. For example, a sandwich assay canbe performed by preparing a biological sample as discussed above, or asis otherwise appropriate for the particular sample, and placing thesample in contact with a solid support on which is immobilized aplurality of capture reagents that bind PDI. The PDI, if present in thesample, binds to the capture reagents. The solid support is thencontacted with detection reagents for PDI. The solid support can bewashed prior to contact with detection reagents to remove unboundreagents. After incubation of the detection reagents for a sufficienttime to bind a substantial portion of the immobilized PDI, any unboundlabeled reagents are removed by, for example, washing. The detectablelabel associated with the detection reagents is then detected. Forexample, in the case of an enzyme used as a detectable label, asubstrate for the enzyme that turns a visible color upon action of theenzyme is placed in contact with the bound detection reagent. A visiblecolor will then be observed in proportion to the amount of the specificantigen in the sample.

[0084] The capture reagent can be any compound that specifically bindsto PDI. Examples of binding moieties that are suitable for use ascapture reagents are described above. One example of a suitable capturereagent is the recombinant polyclonal antibody preparation SCPc.4.PC,which was prepared as described in the Examples. Cells that producethese recombinant polyclonal antibodies were deposited under theBudapest Treaty with the American Type Culture Collection (10801University Boulevard, Manassas, Va. 20110-2209) on ______, and thisdeposit has been assigned ATCC Accession No. ______.

[0085] To immobilize PDI on the solid support, a capture reagent thatspecifically binds to PDI is non-diffusively associated with thesupport. The capture reagents can be non-diffusively immobilized on thesupport either by covalent or non-covalent methods, which are known tothose of skill in the art. See, e.g., Pluskal et al. (1986)BioTechniques 4: 272-283. Suitable supports include, for example,glasses, plastics, polymers, metals, metalloids, ceramics, organics, andthe like. Specific examples include, but are not limited to, microtiterplates, nitrocellulose membranes, nylon membranes, and derivatized nylonmembranes, and also particles, such as agarose, SEPHADEX™, and the like.Assay systems for use in the methods and kits of the invention include,but are not limited to, dipstick-type devices, immunochromatographictest strips and radial partition immunoassay devices, and flow-throughdevices. Conveniently, where the solid support is a membrane, the samplewill flow through the membrane, for example, by gravity, capillaryaction, or under positive or negative pressure.

[0086] Preferred assay systems for use in the kits and methods of theinvention are described in EP 447154. These systems employ an apparatusthat includes a porous member such as a membrane or a filter onto whichis bound a multiplicity of anchor moieties for PDI. The apparatus alsoincludes a non-absorbent member with a textured surface in communicationwith the lower surface of the porous member. The textured surface of thenon-absorbent member can be a grooved surface such as the surface of arecord or it can be composed of channels, such that when the porous andnon-absorbent members are brought into contact with one another anetwork of capillary channels is formed. The capillary network is formedfrom the contact of the porous member with the textured surface of thenon-absorbent member and can be constructed either before or subsequentto the initial contacting of the porous member with a fluid. In someembodiments, the capillary communication between the porous member andthe non-absorbent member favors delaying the transferal of fluid fromthe porous member to the capillary network formed by the porous memberand the textured surface of the non-absorbent member until the volume ofthe added fluid substantially exceeds the void volume of the porousmember. The transferal of fluid from the porous member to the network ofcapillary channels formed by the porous member and the textured surfaceof the non-absorbent member can occur without the use of external means,such as positive external pressure or vacuum, or contact with anabsorbent material. The devices of the present invention can alsoinclude an optional member which is placed in contact with the uppersurface of the porous member and may be used to partition the uppersurface of the device into discrete openings. Such openings can accesseither the porous member or the textured surface of the non-absorbentsecond member. The optional member can in conjunction with thenon-absorbent member compose a fluid receiving zone in which there is nointervening porous member. A fluid receiving zone constructed from thenon-absorbent member and the optional member provides fluid capacity inaddition to that provided by the network of capillary channels createdby the contact of the porous member and the non-absorbent member. Theopenings in the optional member may include a first fluid opening andalso an additional fluid opening. The first fluid opening functions as aportal for the introduction of the first fluid added to the device. Theadditional fluid opening serves as an additional portal through whichadditional fluids may be added to the inventive device.

[0087] To perform an assay using these devices, a volume of the sampleis added to the porous member, where the sample permeates the voidvolume of the porous member and thereby contacts the anchor moietiesimmobilized on the porous member. In a non-competitive assay, the sampleto be assayed is applied to the porous member and the PDI, if present,is bound by the anchor moieties. A detection reagent for PDI is thenadded as an additional fluid; these bind to the complex of PDI andcapture reagent. Alternatively, the detection reagent can be added tothe sample prior to application of the sample to the porous member sothat the binding of detection reagent to PDI occurs prior to the bindingof PDI to the capture reagent. In another embodiment, the capturereagent and detection reagent are added to the sample, after which thecomplex of capture reagent, PDI, and detection reagent binds to abinding agent that is either combined with these reagents or isimmobilized on the porous member. An additional fluid containingreagents to effect a separation of free from bound labeled reagents canbe added to remove excess detection reagent, if needed.

[0088] This device is designed to provide sufficient sensitivity tomeasure low concentrations of PDI because one can use large amounts ofsample and efficiently remove the excess of detection reagent. Indeed,the efficient separation of free from bound label achieved by thenetwork of capillary channels of this device improves the discriminationof specific PDI-associated signal over non-specific background signal.If needed, a signal developer solution is then added to enable the labelof the detection moiety to develop a detectable signal. The signaldeveloped can then be related to the concentration of the target ligandwithin the sample. In a preferred embodiment, the transfer of fluidbetween the porous first member of the device and the network ofcapillary channels formed by the contact of the porous member andtextured surface of the non-absorbent second member of the device isgenerally self-initiated at the point when the total volume of fluidadded to the device exceeds the void volume of the porous member, thusobviating the need for active interaction by the user to remove excessfluid from the analyte detection zone. The point at which the fluidtransfer is initiated is dependent upon the objectives of the assay.Normally, it is desirable to contact the sample with all of the zones onthe porous member which contain immobilized receptor. This methodenables the detection of PDI in a manner that is simple, rapid,convenient, sensitive and efficient in the use of reagents.

[0089] Competitive binding assays can also be used to detect PDI.Conveniently, these assays are performed using the described devices byadding to a sample a labeled analog of PDI. The labeled analog and PDIpresent in the sample compete for the binding sites of the capturereagents. Alternatively, the capture reagents can be combined with thesample and labeled analogs with subsequent immobilization of the capturereagents onto the porous member through contact with a binding agent. Anadditional fluid to separate the free from bound label may be added tothe device, followed if needed by a signal development solution toenable detection of the label of the labeled analog which has complexedwith capture reagent immobilized on the porous member. The amount oflabeled PDI bound to the porous member is related to the concentrationof PDI in the sample.

[0090] This invention also provides kits for the detection and/orquantification of PDI by the described methods. The kits can include acontainer containing one or more of the above-discussed detectionreagents with or without labels, and capture reagents, either free orbound to solid supports. Also included in the kits can be a suitablemembrane, preferably in the form of an assay apparatus that is adaptedto use in the described assay. Preferably, the kits will also includereagents used in the described assays, including reagents useful fordetecting the presence of the detectable labels. Other materials usefulin the performance of the assays can also be included in the kits,including test tubes, transfer pipettes, and the like. The kits can alsoinclude written instructions for the use of one or more of thesereagents in any of the assays described herein.

[0091] The kits of the invention can also include an internal and/or anexternal control. An internal control can consist of the PDIpolypeptide. The control antigen can conveniently be preattached to acapture reagent in a zone of the solid support adjacent to the zone towhich the sample is applied. The external control can also consist ofthe PDI polypeptide. Typically, the antigen present in the externalcontrol will be at a concentration at or above the sensitivity limit ofthe assay means. The external control antigen can be diluted in thesample diluent and assayed in the same manner as would a biologicalsample. Alternatively, the external control PDI polypeptide can be addedto an aliquot of an actual biological sample to determine thesensitivity of the assay. The kits of the present invention can containmaterials sufficient for one assay, or can contain sufficient materialsfor multiple assays.

[0092] The methods, compositions and kits provided by the invention arecapable of detecting PDI with high sensitivity. The assays and kits willdetect PDI when present in a sample at a concentration of about 100ng/ml or less. Preferably, the detection limit for PDI will be about 20ng/ml or less, more preferably about 4 ng/ml or less, and still morepreferably the detection limit for PDI will be about 1 ng/ml or less.

[0093] D. Detection Reagents

[0094] The presence of PDI is generally detected using a detectionreagent that is composed of a binding moiety that specifically binds toPDI. The detection reagents are either directly labeled, i.e., compriseor react to produce a detectable label, or are indirectly labeled, i.e.,bind to a molecule comprising or reacting to produce a detectable label.Labels can be directly attached to or incorporated into the detectionreagent by chemical or recombinant methods.

[0095] In one embodiment, a label is coupled to a molecule, such as anantibody that specifically binds to PDI, through a chemical linker.Linker domains are typically polypeptide sequences, such as poly glysequences of between about 5 and 200 amino acids. In some embodiments,proline residues are incorporated into the linker to prevent theformation of significant secondary structural elements by the linker.Preferred linkers are often flexible amino acid subsequences which aresynthesized as part of a recombinant fusion protein comprising the RNArecognition domain. In one embodiment, the flexible linker is an aminoacid subsequence that includes a proline, such as Gly(x)-Pro-Gly(x)where x is a number between about 3 and about 100. In other embodiments,a chemical linker is used to connect synthetically or recombinantlyproduced recognition and labeling domain subsequences. Such flexiblelinkers are known to persons of skill in the art. For example,poly(ethylene glycol) linkers are available from Shearwater Polymers,Inc. Huntsville, Alabama. These linkers optionally have amide linkages,sulfhydryl linkages, or heterofunctional linkages.

[0096] The detectable labels used in the assays of the presentinvention, which are attached to the detection reagent, can be primarylabels (where the label comprises an element that is detected directlyor that produces a directly detectable element) or secondary labels(where the detected label binds to a primary label, e.g., as is commonin immunological labeling). An introduction to labels, labelingprocedures and detection of labels is found in Polak and Van Noorden(1997) Introduction to Immunocytochemistry, 2nd ed., Springer Verlag, NYand in Haugland (1996) Handbook of Fluorescent Probes and ResearchChemicals, a combined handbook and catalogue Published by MolecularProbes, Inc., Eugene, Oreg. Patents that described the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149; and 4,366,241.

[0097] Primary and secondary labels can include undetected elements aswell as detected elements. Useful primary and secondary labels in thepresent invention can include spectral labels such as green fluorescentprotein, fluorescent dyes (e.g., fluorescein and derivatives such asfluorescein isothiocyanate (FITC) and Oregon Green™, rhodamine andderivatives (e.g., Texas red, tetrarhodimine isothiocynate (TRITC),etc.), digoxigenin, biotin, phycoerythrin, AMCA, CyDyes™, and the like),radiolabels (e.g., ³H, ²⁵I, 35S, ¹⁴C, ³² P, ³³P, etc.), enzymes (e.g.,horse radish peroxidase, alkaline phosphatase etc.), spectralcolorimetric labels such as colloidal gold or colored glass or plastic(e.g. polystyrene, polypropylene, latex, etc.) beads. The label can becoupled directly or indirectly to a component of the detection assay(e.g., the detection reagent) according to methods well known in theart. As indicated above, a wide variety of labels may be used, with thechoice of label depending on sensitivity required, ease of conjugationwith the compound, stability requirements, available instrumentation,and disposal provisions.

[0098] Preferred labels include those that use: 1) chemiluminescence(using horseradish peroxidase and/or alkaline phosphatase withsubstrates that produce photons as breakdown products as describedabove) with kits being available, e.g., from Molecular Probes, Amersham,Boehringer-Mannheim, and Life Technologies/Gibco BRL; 2) colorproduction (using both horseradish peroxidase and/or alkalinephosphatase with substrates that produce a colored precipitate (kitsavailable from Life Technologies/Gibco BRL, and Boehringer-Mannheim));3) fluorescence using, e.g., an enzyme such as alkaline phosphatase,together with the substrate AttoPhos (Amersham) or other substrates thatproduce fluorescent products, 4) fluorescence (e.g., using Cy-5(Amersham), fluorescein, and other fluorescent tags); 5) radioactivity.Other methods for labeling and detection will be readily apparent to oneskilled in the art.

[0099] For use of the present invention in the clinic, preferred labelsare non-radioactive and readily detected without the necessity ofsophisticated instrumentation. Preferably, detection of the labels willyield a visible signal that is immediately discemable upon visualinspection. One preferred example of detectable secondary labelingstrategies uses an antibody that recognizes PDI in which the antibody islinked to an enzyme (typically by recombinant or covalent chemicalbonding). The antibody is detected when the enzyme reacts with itssubstrate, producing a detectable product. Preferred enzymes that can beconjugated to detection reagents of the invention include, e.g.,β-galactosidase, luciferase, horse radish peroxidase, and alkalinephosphatase. The chemiluminescent substrate for luciferase is luciferin.One embodiment of a fluorescent substrate for β-galactosidase is4-methylumbelliferyl-β-D-galactoside. Embodiments of alkalinephosphatase substrates include p-nitrophenyl phosphate (pNPP), which isdetected with a spectrophotometer; 5-bromo-4-chloro-3-indolylphosphate/nitro blue tetrazolium (BCIP/NBT) and fast red/napthol AS-TRphosphate, which are detected visually; and4-methoxy-4-(3-phosphonophenyl) spiro[1,2-dioxetane-3,2′-adamantane],which is detected with a luminometer. Embodiments of horse radishperoxidase substrates include 2,2′azino-bis(3-ethylbenzthiazoline-6sulfonic acid) (ABTS), 5-aminosalicylic acid (5AS), o-dianisidine, ando-phenylenediamine (OPD), which are detected with a spectrophotometer;and 3,3,5,5′-tetramethylbenzidine (TMB), 3,3'diaminobenzidine (DAB),3-amino-9-ethylcarbazole (AEC), and 4-chloro-1-naphthol (4CIN), whichare detected visually. Other suitable substrates are known to thoseskilled in the art. The enzyme-substrate reaction and product detectionare performed according to standard procedures known to those skilled inthe art and kits for performing enzyme immunoassays are available asdescribed above.

[0100] The presence of a label can be detected by inspection, or adetector which monitors a particular probe or probe combination is usedto detect the detection reagent label. Typical detectors includespectrophotometers, phototubes and photodiodes, microscopes,scintillation counters, cameras, film and the like, as well ascombinations thereof. Examples of suitable detectors are widelyavailable from a variety of commercial sources known to persons ofskill. Commonly, an optical image of a substrate comprising boundlabeling moieties is digitized for subsequent computer analysis.

EXAMPLES

[0101] The following examples are offered to illustrate, but not tolimit the present invention.

Example 1 Synthesis and Screening of a Cryptosporidium parvum Mixed cDNALibrary

[0102] This Example describes the cloning of cDNAs that encode theprotein disulfide isomerase antigen of C. parvum.

[0103] A. Culture of C. Parvum and Preparation of Soluble Antigen

[0104]Cryptosporidium parvum oocysts were obtained from the TuftsUniversity School of Veterinary Medicine. Organisms were harvested andwashed three times in 0.01M phosphate buffered saline (PBS), pH 7.6. Thecell pellet was resuspended in 1 ml of PBS and subjected to 4 cycles offlash-freezing and thawing. Cryptosporidium oocysts were sonicated for12 min using a VirSonic 475 Ultrasonic Cell Disrupter. Cell disruptionwas monitored by microscopic inspection. Cells and debris were removedby centrifugation at 14,000× g for 20 min at 4° C. The supernatantcontaining soluble antigen was transferred to a fresh tube, assayed forprotein content, and used for immunizations.

[0105] Freshly harvested C. parvum oocysts from bovine feces (originallyof human origin) were obtained from Utah State University, Logan, Utahand Turts University, Boston, Mass. Excystation of sporozoites wasperformed by the method of Yang et al. (1996) Infect. Immun. 64:349-354. Sporozoites were cultured in MDCK cells grown in a 5% CO₂environment at 37° C. Organisms were harvested from MDCK cell culturesupernatants by an initial centrifugation for 10 min at 500× g to removedetached cells and large cellular debris, followed by centrifugation for20 min at 10,000× g. The pellet was washed three times in sterile PBS.

[0106] The entire life cycle of C. parvum was reproduced by infecting amonolayer of bovine fallopian tube epithelial cells (BFTE) in 25 cm²tissue culture flasks with approximately 10⁴ sporozoites and incubating24-72 hr at 37° C. in a candle jar environment (Yang et al., supra.).The organisms from the BFTE cell culture supernatant were purified bypassage through a 3 mm pore size polycarbonate filter (Millipore Corp,Bedford, Mass.). This filtration step permitted the passage of oocystsand sporozoites while retaining cellular debris. Organisms wereconcentrated by an initial centrifugation for 20 min at 10,000× g. Thepellet was then washed three times in sterile PBS. The pellet containingorganisms representing all forms of the C. parvum life cycle was pooledwith the sporozoite pellet purified from the MDCK cell culture above andused immediately for the isolation of total RNA.

[0107] B. Isolation and Purification of RNA from a Cryptosporidiumparvum Mixed Culture

[0108]Cryptosporidium parvum organisms representing all phases of itslife cycle (oocysts and sporozoites) were cultured and harvested asdescribed above. Working quickly, 1.0 ml of solution D (25.0 g guanidinethiocyanate (Boehringer Mannheim, Indianapolis, Ind.), 29.3 ml sterilewater, 1.76 ml 0.75M sodium citrate (pH 7.0), 2.64 ml 10% sarkosyl(Fisher Scientific, Pittsburgh, Pa.), 0.36 ml 2-mercaptoethanol (FisherScientific, Pittsburgh, Pa.)) was added to the pellet while vortexing.The cell suspension was pulled through an 18-gauge needle until viscousand all cells were lysed, after which the suspension was transferred toa microcentrifuge tube. The suspension was then pulled through a22-gauge needle followed by a 25-gauge needle an additional 5-10 times.

[0109] The sample was divided evenly between two microcentrifuge tubesand the following added in order, with mixing by inversion after eachaddition: 100 μl 2M sodium acetate (pH 4.0), 1.0 ml water saturatedphenol (Fisher Scientific, Pittsburgh, Pa.): 200μl chloroform/isoamylalcohol 49:1 (Fisher Scientific, Pittsburgh, Pa.). The solution wasvortexed for 10 seconds and incubated on ice for 15 minutes. Followingcentrifugation (10,000 g) for 20 minutes at 2-8° C., the aqueous phasewas transferred to a fresh tube. An equal volume of water saturatedphenol:chloroform:isoamyl alcohol (50:49:1) was added, and the tube wasvortexed for ten seconds. After a 15 min incubation on ice, the samplewas centrifuged for 20 minutes at 2-8° C., and the aqueous phase wastransferred to a fresh tube and precipitated with an equal volume ofisopropanol at −20° C. for a minimum of 30 minutes. Followingcentrifugation (10,000 g) for 20 minutes at 4° C., the supernatant wasaspirated away, the tubes briefly spun and all traces of liquid removed.

[0110] The RNA pellets were each dissolved in 300 μl of solution D,combined, and precipitated with an equal volume of isopropanol at −20°C. for a minimum of 30 minutes. The sample was centrifuged (10,000 g)for 20 minutes at 4° C., the supernatant aspirated as before, and thesample rinsed with 100 μl of ice-cold 70% ethanol. The sample was againcentrifuged (10,000 g) for 20 minutes at 4° C., the 70% ethanol solutionaspirated, and the RNA pellet dried in vacuo. The pellet was resuspendedin 100 μl of sterile distilled water. The concentration was determinedby A₂₆₀ using an absorbance of 1.0 for a concentration of 40 μg/ml. TheRNAs were stored at −80° C.

[0111] Messenger RNA (MRNA) was purified from total RNA using anOligotex Mini-Kit™ MRNA isolation kit (Qiagen, Santa Clarita, Calif.)according to the manufacturer's recommendations. The concentration ofMRNA was determined by A₂₆₀ using an absorbance of 1.0 for aconcentration of 40μg/ml. The mRNAs were stored at −80° C.

[0112] C. Synthesis of Lambda cDNA Libraries.

[0113] The mRNAs (5.0 μg) purified above were used to synthesize thefirst and second strands of cDNA using a cDNA synthesis kit (Stratagene,San Diego, Calif.) following the manufacturer's recommendations. Theresulting cDNA was selected for inserts greater than 500 base pairs inlength. The size-selected cDNA was then ligated into the Uni-ZAP XRvector (Stratagene, San Diego, Calif.) and packaged with Gigapak Goldpackaging extract (Stratagene, San Diego, Calif.) followingmanufacturer's recommendations. The primary sizes for the mixed librarywere determined by plating serial dilutions of the packaged library (seebelow) to be 1.4×10⁶ plaque-forming units (pfu). Background wasdetermined to be <2.0% through blue/white selection (see below). Theresulting Uni-ZAP XR™ lambda phage library was amplified once beforescreening to ensure stability of the library, titered, and stored at 4°C.

[0114] D. Plating Lambda Phage CDNA Library.

[0115] Starting with a lambda phage stock, a series of 100-folddilutions (10 l to 1.0 ml) were made in SM buffer (Stratagene, SanDiego, Calif.). The diluted phage samples (10μl) were added to 200 μl ofan overnight culture of Escherichia coli strain XL1-Blue MRF′(Stratagene, San Diego, Calif.) adjusted to OD₆₀₀=0.5 in 10 mM MgSO₄ insterile 15 ml tubes and incubated at 37° C. for 15 min. After adding 3.0ml of NZY top-agar at 55° C., the mixture was poured and evenlydistributed on an NZY agar plate (100 mm) that had been pre-warmed (37°C.-55° C.) to remove any excess moisture on the agar surface. The plateswere cooled to room temperature, at which time the top-agar solidified,and the plates were then inverted and placed at 37° C. For titeringpurposes, the plates were left at 37° C. overnight and the number ofplaques counted and a titer determined.

[0116] In order to determine the background for the library (thepercentage of clones not carrying an insert), several hundred plaqueswere plated as described above. Prior to plating, 15 μl of 0.5Misopropyl-β-D-thiogalactoside (IPTG) and 50 μl of5-bromo-4-chloro-3-indoyl-β-D-galctopyranoside (X-gal) [250 mg/ml (indimethylformamide)] was added to the NZY top agar. These plates wereincubated at 37° C. for 6-8 hours and transferred to room temperatureovernight. Plaques that stained blue correspond to clones that do nothave an insert, while non-staining, white plaques contain an insert. Thepercentage of background plaques was calculated by dividing the numberof blue plaques by the total number of plaques.

[0117] E. Screening of C. parvum Mixed CDNA Libraries with MonoclonalAntibody CP.2

[0118] The C. parvum mixed cDNA library was plated, separately, on large(150 mm) NZY agar plates at a density of approximately 10,000-20,000pfu/plate as described above, except that 600 μl of OD₆₀₀=0.5 XL1-Bluecells and nine ml of NZY top agar were used for plating. When theplaques reached 0.5-1.0 mm in diameter (4-5 hr), nitrocellulose filterlifts (diameter 137 mm, pore size 0.45 μm, BA85 Protran, Schleicher andSchuell, Keene, N.H.) soaked in 10 mM isopropyl-β-D-thiogalactoside(IPTG) were placed on the agar plates, marked asymmetrically with aneedle, and placed at 20° C.

[0119] After overnight incubation, the filters were carefully removedfrom the plates with membrane forceps, rinsed briefly in TBST (40 mMTRIS, 150 mM NaCl, 0.05% Tween 20 (Fisher Chemical, Pittsburgh, Pa.), pH7.5) to remove any debris from the lifts, and incubated for greater thanone hour in block (1% BSA solution containing 20 mM Tris, 150 mM NaCl,and 0.1% sodium azide, pH 8.0). The filters were then incubated inCP.2-alkaline phosphatase (AP) conjugate (Example 19A) at 2.5 μg/ml, inblock, for a minimum of 4 hours. The filters were washed three timeswith TBST for 5 min each. After the final wash, the filters weredeveloped as described in Example 13.

[0120] The filters were aligned with the agar plates through theasymmetric needle marks and plaques individually cored from the agarplates and transferred to 250-500 μl of SM buffer. The plaques werechosen based on their staining intensity with CP.2-AP conjugate, rangingfrom light staining to dark staining. These plaques were purified tohomogeneity through iterative rounds of the plating/filter liftprocedure described above.

[0121] The DNA inserts were subcloned ‘rescued’ into the plasmid vectorpBluescript (Stratagene, San Diego, Calif.) through an in vivo excisionprocess following the manufacturer's recommendations. The DNA sequenceat the 3′ end of each clone was determined by the dideoxy chaintermination method using Sequenase II™ DNA cloning kit (U.S.Biochemical) and an oligonucleotide, primer A (Table 1), that binds tothe DNA sequence on the 3′ side of the insert in the pBluescript vector.One clone was sequenced. The polynucleotide sequence of the cDNA (SEQ IDNO: 1) and the deduced amino acid sequence (SEQ ID NO: 2) are presentedin FIG. 3A and FIG. 3B, respectively.

Example 2 Cloning of the Cryptosporidium parvum Protein DisulfideIsomerase cDNA

[0122] PCR primers were made corresponding to the coding sequence at the5′ and 3′ ends of the C. parvum PDI, primers B and C, respectively(Table 1). The primers were based on a sequence found in the literature(Blunt et al., supra.). In addition, the 5′ primer contains 20 basepairs of vector sequence at its 5′ -end corresponding to the 3′-end ofthe pBRnsiH3 vector (described in copending, commonly assigned U.S.patent application Ser. No. 08/835,159, filed Apr. 4, 1997). The 3′primer contains the 19 base pairs of the tet promoter removed by HindIIIdigestion, in addition to 20 base pairs of vector sequence 3′ to theHindIII site at its 5′ end (see, Example 18 of U.S. patent applicationSer. No. 08/835,159, filed Apr. 4, 1997).

[0123] The PDI insert was amplified with the primers described above and1 μl (˜50 ng) of C. parvum genomic DNA as template per reaction. Theamplification (3×100 μl reactions) was performed using Expand™ DNApolymerase and the reactions pooled and purified as described in Example19 of U.S. patent application Ser. No. 08/835,159. The PDI insert (10ng) was annealed with the pBRnsiH3 (10 ng) at a 3:1 molar excess ofinsert to vector, and an aliquot electroporated into 40 μl ofelectrocompetent E. coli strain, DH10 B as described in Example 9. Thetransformed cells were diluted to 1.0 ml with 2× YT broth and 10 μl, 100μl and 300 μl plated on LB agar plates supplemented with tetracycline(10 g/ml) and grown overnight at 37° C. Four colonies were picked into 3ml 2× YT supplemented tetracycline (10 μg/ml) and grown overnight at 37°C. The following day, glycerol freezer stocks were made for long termstorage at −80° C.

[0124] These four clones were sequenced by the dideoxy chain terminationmethod using a Sequenase II™ DNA cloning kit (U.S. Biochemical) andoligonucleotide primers D-L (Table 1). Primers D-J bind to the proteindisulfide isomerase DNA sequence and primers K and L (Table 1) bind onthe 5′ and 3′ side of the insert in the pBR vector, respectively. Thenucleotide sequence of the PDI cDNA (SEQ ID NO: 1), and thecorresponding deduced amino acid sequence (SEQ ID NO: 2), are presentedin FIG. 3A and FIG. 3B, respectively.

[0125] A search of the cDNA polynucleotide sequence against the NationalCenter for Biotechnology Information (NCBI) non-redundant nucleotidedatabase using the BLAST search engine revealed that the clone has adeduced amino acid sequence similar to that of Cryptosporidium parvumprotein disulfide isomerase (PDI) (Blunt et al. (1996) Gene 181:221-223). Two significant differences were found in the four clonesisolated as described herein as compared to the Blunt et al. sequence.The first was a single base insertion (G) between positions 1065 and1066 (numbering based on Genbank Accession No. U48261). The second wasthe deletion of a single base (C) at position 1161 (numbering based onGenbank Accession No. U48261). These changes resulted in a frame-shiftspanning 32 amino acids.

[0126] A comparison of the Cryptosporidium parvum protein disulfideisomerase protein sequence as described herein to that of the previouslyreported amino acid sequence for C. parvum PDI (Blunt et al. (1996) Gene181: 221-223) revealed a 32 amino acid region in which the amino acidsequences diverge (shown below). The numbering at the beginning andending of the sequence corresponds to the numbering convention used byBlunt et al. The 32 amino acid region that differs between the twosequences is highlighted in bold type.

[0127] Applicants 250 SREGYTAWFCGTNEDFAKYASNIRKVAADYREKYAFVFLDT 290 (SEQID NO: 3)

[0128] Blunt et al. 250 SREGYTPGSVVLTRTSPSMLQTLERLQLITEKSMPLFSLDT 290(SEQ ID NO: 4)

[0129] The nucleotide sequence of the region of Applicants' PDI sequencethat differs from that of Blunt et al. is: shown in FIG. 3A.

[0130] The inventors believe that PDI sequence described herein is thecorrect sequence based on the following: 1) the identical insertion anddeletion occurred in four clones from two independent cloningexperiments; 2) the frame-shifted sequence described herein shares agreater percent identify to protein disulfide isomerase from otherorganisms than does the same region from the literature. TheCryptosporidium parvum protein disulfide isomerase antigen was expressedand purified as described in Example 15. TABLE 1 PCR and SequencingPrimer Sequences A: 5′-GTAAAACGACGGCCAGTGAATTG-3′ (SEQ ID NO:6) B:5′-ACCCGTTTTTTTGGATGGAGTGAAACG (SEQ ID NO:7)ATGATCGGAATTCGTAGCTTGGTTTCA-3′ C:5′-GTGATAAACTACCGCATTAAAGCTTATCGATGATAAGCTGTCAAT (SEQ ID NO:8)TAGTGATGGTGATGGTGATGGAGTTCGTCACGAGATTCCTTTTC-3′ D:5′-TCCAAGGTAGTCTTGGGTGG-3′ (SEQ ID NO:9) E: 5′-AAGCTCTCTCCCCCAGTACA-3′(SEQ ID NO:10) F: 5′-GCAGTCCAAGTTGCTGAATC-3′ (SEQ ID NO:11) G:5′-CTCGAGGTATTACACAAGGA-3′ (SEQ ID NO:12) H: 5′-CCAAGTATGCTTCAAACATT-3′(SEQ ID NO:13) I: 5′-TTCGACTCTGTTGAGCCATT-3′ (SEQ ID NO:14) J:5′-TGTGGACACTGTAAGAACCTC-3′ (SEQ ID NO:15) K: 5′-GAGGATGACGATGAGCGC-3′(SEQ ID NO:16) L: 5′-GCAACTCTCTACTGTTTCTCC-3′ (SEQ ID NO:17) M:5′-TCGCTGCCCAACCAGCCATG-3′ (SEQ ID NO:18) N:5′-GTGATAAACTACCGCATTAAAGCTTATCGATGATAAGC (SEQ ID NO:19)TGTCAATTAGTGATGGTGATGGTGATGACAATCCCTG-3′

Example 3 Immunization of Mice with Crude Cryptosporidium SolubleAntigen and Purification of RNA from Mouse Spleens

[0131] Mice were immunized by the following method based on experienceof the timing of spleen harvest for optimal recovery of mRNA coding forantibody. Two species of mice were used: Balb/c (Charles RiverLaboratories, Wilmington, Mass.) and A/J (Jackson Laboratories, BarHarbor, Me.). Mice were immunized intraperitoneally or subcutaneouslywith C. parvum soluble antigen (Example 1A) using 50 μg protein inFreund's complete adjuvant on day 0, and with 100 μg antigen on day 28.Tests bleeds of mice were obtained through puncture of the retro-orbitalsinus. If, by testing the titers, they were deemed high by ELISA usingbiotinylated antigen immobilized via streptavidin, the mice were boostedwith 100 μg of protein on day 70, 71 and 72, with subsequent sacrificeand splenectomy on day 77. If titers of antibody were not deemedsatisfactory, mice were boosted with 100 μg antigen on day 56 and a testbleed taken on day 63. If satisfactory titers were obtained, the animalswere boosted with 100 μg of antigen on day 98, 99, and 100 and thespleens harvested on day 105.

[0132] The spleens were harvested in a laminar flow hood and transferredto a petri dish, trimming off and discarding fat and connective tissue.The spleens were, working quickly, macerated with the plunger from asterile 5 cc syringe in the presence of 1.0 ml of solution D (25.0 gguanidine thiocyanate (Boehringer Mannheim, Indianapolis, Ind.), 29.3 mlsterile water, 1.76 ml 0.75 M sodium citrate (pH 7.0), 2.64 ml 10%sarkosyl (Fisher Scientific, Pittsburgh, Pa.), 0.36 ml 2-mercaptoethanol(Fisher Scientific, Pittsburgh, Pa.)). The spleen suspension was pulledthrough an 18 gauge needle until viscous and all cells were lysed, thentransferred to a microcentrifuge tube. The petri dish was washed with100 μl of solution D to recover any remaining spleen, and this wastransferred to the tube. The suspension was then pulled through a 22gauge needle an additional 5-10 times.

[0133] The sample was divided evenly between two microcentrifuge tubesand the following added, in order, with mixing by inversion after eachaddition: 100 μl 2 M sodium acetate (pH 4.0), 1.0 ml water-saturatedphenol (Fisher Scientific, Pittsburgh, Pa.):200 μl chloroform/isoamylalcohol 49:1 (Fisher Scientific, Pittsburgh, Pa.). The solution wasvortexed for 10 seconds and incubated on ice for 15 min. Followingcentrifugation at 14 krpm for 20 min at 2-8° C., the aqueous phase wastransferred to a fresh tube. An equal volume of water saturatedphenol:chloroform:isoamyl alcohol (50:49:1) was added, and the tube wasvortexed for ten seconds. After a 15 min incubation on ice, the samplewas centrifuged for 20 min at 2-8° C., and the aqueous phase wastransferred to a fresh tube and precipitated with an equal volume ofisopropanol at −20° C. for a minimum of 30 min. Following centrifugationat 14 krpm for 20 min at 4° C., the supernatant was aspirated away, thetubes briefly spun and all traces of liquid removed.

[0134] The RNA pellets were each dissolved in 300 μl of solution D,combined, and precipitated with an equal volume of isopropanol at −20°C. for a minimum of 30 min. The sample was centrifuged 14 krpm for 20min at 4° C., the supernatant aspirated as before, and the sample rinsedwith 100 μl of ice-cold 70% ethanol. The sample was again centrifuged 14krpm for 20 min at 4° C., the 70% ethanol solution aspirated, and theRNA pellet dried in vacuo. The pellet was resuspended in 100 μl ofsterile distilled water. The concentration was determined by A₂₆₀ usingan absorbance of 1.0 for a concentration of 33 μg/ml. The RNAs werestored at −80° C.

Example 4 Preparation of Complementary DNA (CDNA)

[0135] The total RNA purified from mouse spleens as described above wasused directly as template for cDNA preparation. RNA (50 μg) was dilutedto 100 μL with sterile water, and 10 μL of 130 ng/μL oligo dT₁₂(synthesized on Applied Biosystems Model 392 DNA synthesizer) was added.The sample was heated for 10 min at 70° C., then cooled on ice. Forty μL5× first strand buffer was added (Gibco/BRL, Gaithersburg, Md.), alongwith 20 μL 0.1 M dithiothreitol (Gibco/BRL, Gaithersburg, Md.), 10 μL 20mM deoxynucleoside triphosphates (dNTP's, Boehringer Mannheim,Indianapolis, IN), and 10 μL water on ice. The sample was then incubatedat 37° C. for 2 min. Ten μL reverse transcriptase (Superscript™ II,Gibco/BRL, Gaithersburg, Md.) was added and incubation was continued at37° C. for 1 hr. The cDNA products were used directly for polymerasechain reaction (PCR).

Example 5 Amplification of cDNA by PCR

[0136] To amplify substantially all of the H and L chain genes usingPCR, primers were chosen that corresponded to substantially allpublished sequences. Because the nucleotide sequences of the aminoterminals of H and L contain considerable diversity, 33 oligonucleotideswere synthesized to serve as 5′ primers for the H chains, and 29oligonucleotides were synthesized to serve as 5′ primers for the kappa Lchains as described in co-pending, commonly assigned U.S. patentapplication Ser. No. 08/835,159, filed Apr. 4, 1997. The constant regionnucleotide sequences required only one 3′ primer each to the H chainsand the kappa L chains. Id.

[0137] A 50 μL reaction was performed for each primer pair with 50 pmolof 5′ primer, 50 pmol of 3′ primer, 0.25 μL Taq DNA Polymerase (5units/μL, Boehringer Mannheim, Indianapolis, Ind.), 3 μL cDNA (preparedas described in Example 4), 5 μL 2 mM dNTP's, 5 μL 10× Taq DNApolymerase buffer with MgCl₂ (Boehringer Mannheim, Indianapolis, Ind.),and H₂O to 50 μL. Amplification was done using a GeneAmp® 9600 thermalcycler (Perkin Elmer, Foster City, Calif.) with the following program:94° C. for 1 min; 30 cycles of 94° C. for 20 sec, 55° C. for 30 sec, and72° C. for 30 sec; 72° C. for 6 min; 4° C.

[0138] The dsDNA products of the PCR process were then subjected toasymmetric PCR using only a 3′ primer to generate substantially only theanti-sense strand of the target genes. A 100 μL reaction was done foreach dsDNA product with 200 pmol of 3′ primer, 2 μL of ds-DNA product,0.5 μL Taq DNA Polymerase, 10 μL 2 mM dNTP's, 10 μL 10× Taq DNApolymerase buffer with MgCl₂ (Boehringer Mannheim, Indianapolis, Ind.),and H₂O to 100 μL. The same PCR program as that described above was usedto amplify the single-stranded (ss)-DNA.

Example 6 Purification of ss-DNA by High Performance LiquidChromatography and Kinasing ss-DNA

[0139] The H chain ss-PCR products and the L chain ss-PCR products wereethanol precipitated by adding 2.5 volumes ethanol and 0.2 volumes 7.5 Mammonium acetate and incubating at −20° C. for at least 30 min. The DNAwas pelleted by centrifuging in an Eppendorf centrifuge at 14 krpm for10 min at 2-8° C. The supernatant was carefully aspirated, and the tubeswere briefly spun a 2nd time. The last drop of supernatant was removedwith a pipette. The DNA was dried in vacuo for 10 min on medium heat.The H chain products were pooled in 210 μL water and the L chainproducts were pooled separately in 210 μL water. The ss-DNA was purifiedby high performance liquid chromatography (HPLC) using a Hewlett Packard1090 HPLC and a Gen-Pak™ FAX anion exchange column (Millipore Corp.,Milford, Mass.). The gradient used to purify the ss-DNA is shown inTable 2, and the oven temperature was at 60° C. Absorbance was monitoredat 260 nm. The ss-DNA eluted from the HPLC was collected in 0.5 minfractions. Fractions containing ss-DNA were ethanol precipitated,pelleted and dried as described above. The dried DNA pellets were pooledin 200 μL sterile water. TABLE 2 HPLC gradient for purification ofss-DNA Time (min) % A % B % C Flow (ml/min) 0 70 30 0 0.75 2 40 60 00.75 32 15 85 0 0.75 35 0 100 0 0.75 40 0 100 0 0.75 41 0 0 100 0.75 450 0 100 0.75 46 0 100 0 0.75 51 0 100 0 0.75 52 70 30 0 0.75

[0140] Buffer A is 25 mM Tris, 1 mM EDTA, pH 8.0

[0141] Buffer B is 25 mM Tris, 1 mM EDTA, 1 M NaCl, pH 8.0

[0142] Buffer C is 40 mm phosphoric acid

[0143] The ss-DNA was phosphorylated on the 5′ end in preparation formutagenesis (Example 8). Twenty-four μL 10× kinase buffer (United StatesBiochemical, Cleveland, Ohio), 10.4 μL 10 mM adenosine-5′-triphosphate(Boehringer Manrheim, Indianapolis, Ind.), and 2 μL polynucleotidekinase (30 units/μL, United States Biochemical, Cleveland, Ohio) wasadded to each sample, and the tubes were incubated at 37° C. for 1 hr.The reactions were stopped by incubating the tubes at 70° C. for 10 min.The DNA was purified with one extraction of equilibrated phenol(pH >8.0, United States Biochemical, Cleveland, Ohio):chloroform:isoamylalcohol (50:49:1) and one extraction with chloroform:isoamyl alcohol(49: 1). After the extractions, the DNA was ethanol precipitated andpelleted as described above. The DNA pellets were dried, then dissolvedin 50 μL sterile water. The concentration was determined by measuringthe absorbance of an aliquot of the DNA at 260 n using 33 μg/ml for anabsorbance of 1.0. Samples were stored at −20° C.

Example 7 Preparation of Uracil Templates used in Generation of SpleenAntibody Phage Libraries

[0144] One ml of E. coli CJ236 (BioRAD, Hercules, Calif.) overnightculture was added to 50 ml 2× YT in a 250 ml baffled shake flask. Theculture was grown at 37° C. to OD₆₀₀=0.6, inoculated with 10 μl of a{fraction (1/100)} dilution of BS45 vector phage stock (described inco-pending, commonly assigned U.S. patent application Ser. No.08/835,159, filed Apr. 4, 1997) and growth continued for 6 hr.Approximately 40 ml of the culture was centrifuged at 12 krpm for 15minutes at 4° C. The supernatant (30 ml) was transferred to a freshcentrifuge tube and incubated at room temperature for 15 minutes afterthe addition of 15 μl of 10 mg/ml RNaseA (Boehringer Mannheim,Indianapolis, Ind.). The phage were precipitated by the addition of 7.5ml of 20% polyethylene glycol 8000 (Fisher Scientific, Pittsburgh,Pa.)/3.5M ammonium acetate (Sigma Chemical Co., St. Louis, Mo.) andincubation on ice for 30 min. The sample was centrifuged at 12 krpm for15 min at 2-8° C. The supernatant was carefully discarded, and the tubewas briefly spun to remove all traces of supernatant. The pellet wasresuspended in 400 μl of high salt buffer (300 mM NaCl, 100 mM Tris pH8.0, 1 mM EDTA), and transferred to a 1.5 ml tube.

[0145] The phage stock was extracted repeatedly with an equal volume ofequilibrated phenol:chloroform:isoamyl alcohol (50:49:1) until no traceof a white interface was visible, and then extracted with an equalvolume of chloroform:isoamyl alcohol (49:1). The DNA was precipitatedwith 2.5 volumes of ethanol and ⅕ volume 7.5 M ammonium acetate andincubated 30 min at −20° C. The DNA was centrifuged at 14 krpm for 10min at 4° C., the pellet washed once with cold 70% ethanol, and dried invacuo. The uracil template DNA was dissolved in 30 μl sterile water andthe concentration determined by A₂₆₀ using an absorbance of 1.0 for aconcentration of 40 μg/ml. The template was diluted to 250 ng/μl withsterile water, aliquoted, and stored at −20° C.

Example 8 Mutagenesis of Uracil Template with ss-DNA and Electroporationinto E. coli to Generate Antibody Phage Libraries

[0146] Antibody phage display libraries were generated by simultaneouslyintroducing single-stranded heavy and light chain genes onto a phagedisplay vector uracil template. A typical mutagenesis was performed on a2 μg scale by mixing the following in a 0.2 ml PCR reaction tube: 8 μlof (250 ng/μl) uracil template (Example 7), 8 μl of 10× annealing buffer(200 mM Tris pH 7.0, 20 mM MgCl₂, 500 mM NaCl), 3.33 μl of kinasedsingle-stranded heavy chain insert (100 ng/μl), 3.1 μl of kinasedsingle-stranded light chain insert (100 ng/μl), and sterile water to 80μl. DNA was annealed in a GeneAmp™ 9600 thermal cycler using thefollowing thermal profile: 20 sec at 94° C., 85° C. for 60 sec, 85° C.to 55° C. ramp over 30 min, hold at 55° C. for 15 min. The DNA wastransferred to ice after the program finished. The extension/ligationwas carried out by adding 8 μl of 10× synthesis buffer (5 mM each dNTP,10 mM ATP, 100 mM Tris pH 7.4, 50 mM MgCl₂, 20 mM DTT), 8 μl T4 DNAligase (1 U/μl, Boehringer Mannheim, Indianapolis, Ind.), 8 μl dilutedT7 DNA polymerase (1 U/μl, New England BioLabs, Beverly, Mass.) andincubating at 37° C. for 30 min. The reaction was stopped with 300 μl ofmutagenesis stop buffer (10 mM Tris pH 8.0, 10 mM EDTA).

[0147] The mutagenesis DNA was extracted once with equilibrated phenol(pH>8):chloroform:isoamyl alcohol (50:49:1), once withchloroform:isoamyl alcohol (49:1), and the DNA was ethanol precipitatedat −20° C. for at least 30 min. The DNA was pelleted and the supernatantcarefully removed as described above. The sample was briefly spun againand all traces of ethanol removed with a pipetman. The pellet was driedin vacuo. The DNA was resuspended in 4 μl of sterile water.

[0148] One μl mutagenesis DNA (500 ng) was transferred into 40 μlelectrocompetentE. coli DH12S (Gibco/BRL, Gaithersburg, Md.) using theelectroporation conditions in Example 9. The transformed cells weremixed with 1.0 ml 2 × YT broth (Sambrook et al., supra) and transferredto 15 ml sterile culture tubes. The first round antibody phage was madeby shaking the cultures overnight at 23° C. and 300 rpm. The efficiencyof the electroporation was measured by plating 10 μl of 10⁻³ and 10⁻⁴dilutions of the cultures on LB agar plates (see Example 12). Theseplates were incubated overnight at 37° C. The efficiency was determinedby multiplying the number of plaques on the 10⁻³ dilution plate by 10⁵or multiplying the number of plaques on the 10⁻⁴ dilution plate by 10⁶.The overnight cultures from the electroporations were transferred to 1.5ml tubes, and the cells were pelleted by centrifuging at 14 krpm for 5min. The supernatant, which is the first round of antibody phage, wasthen transferred to 15 ml sterile centrifuge tubes with plug seal caps.

Example 9 Transformation of E. coli by Electroporation

[0149] The electrocompetent E. coli cells were thawed on ice. DNA wasmixed with 20-40 μL electrocompetent cells by gently pipetting the cellsup and down 2-3 times, being careful not to introduce an air bubble. Thecells were transferred to a Gene Pulser cuvette (0.2 cm gap, BioRAD,Hercules, Calif.) that had been cooled on ice, again being careful notto introduce an air bubble in the transfer. The cuvette was placed inthe E. coli Pulser (BioRAD, Hercules, Calif.) and electroporated withthe voltage set at 1.88 kV according to the manufacturer'srecommendation. The transformed sample was immediately diluted to 1 mlwith 2× YT broth and processed as procedures dictated.

Example 10 Preparation of Biotinylated Antigens and BiotinylatedAntibodies

[0150] Crude Cryptosporidium soluble antigen (1 mg/mL, Example 1A) wasextensively dialyzed into BBS. After dialysis, the protein was reactedwith biotin-XX-NHS ester (Molecular Probes, Eugene, Orrg., stocksolution at 40 mM in dimethylformamide) at a final concentration of 0.5mM. The reaction was incubated at room temperature for 90 min. After 90min, the protein was dialyzed extensively against BBS at 2-8° C. Afterdialysis, the biotin concentration was 5×10⁻⁵ M. The biotinconcentration can be measured using the HABA reagent kit (Pierce,Rockford, Ill.). The biotinylated crude Cryptosporidium soluble antigenwas stored at −80° C.

[0151] Antibodies were reacted with 3-(N-maleimidylpropionyl)biocytinusing a free cysteine at the carboxy terminus of the heavy chain. Thecysteine was reduced by adding DTT to a final concentration of 1 mM for30 min at room temperature. The antibody was passed through a SephadexG50 desalting column equilibrated in column buffer.3-(N-maleimidylpropionyl)biocytin was added to a final concentration of1 mM. Reactions were allowed to proceed at room temperature for 60 min.Antibodies were dialyzed extensively into BBS and stored at 2-8° C.

Example 11 Preparation of Avidin Magnetic Latex

[0152] The magnetic latex (Estapor, 10% solids, Bangs Laboratories,Fishers, Ind.) was thoroughly resuspended and 2 ml aliquoted into a 15ml conical tube. The magnetic latex was suspended in 12 ml distilledwater and separated from the solution for 10 min using a magnet(PerSeptive Biosystems, Framingham Mass.). While maintaining theseparation of the magnetic latex with the magnet, the liquid wascarefully removed using a 10 ml sterile pipette. This washing processwas repeated an additional three times. After the final wash, the latexwas resuspended in 2 ml of distilled water. In a separate 50 ml conicaltube, 10 mg of avidin-HS (NeutrAvidin, Pierce, Rockford, Ill.) wasdissolved in 18 ml of 40 mM Tris, 0.15 M sodium chloride, pH 7.5 (TBS).While vortexing, the 2 ml of washed magnetic latex was added to thediluted avidin-HS and the mixture vortexed an additional 30 seconds.This mixture was incubated at 45° C. for 2 hr, shaking every 30 minutes.The avidin magnetic latex was separated from the solution using a magnetand washed three times with 20 ml BBS as described above. After thefinal wash, the latex was resuspended in 10 ml BBS and stored at 4° C.

[0153] Immediately prior to use, the avidin magnetic latex wasequilibrated in panning buffer (40 mM TRIS, 150 mM NaCl, 20 mg/ml BSA,0.1% Tween 20 (Fisher Scientific, Pittsburgh, Pa.), pH 7.5). The avidinmagnetic latex needed for a panning experiment (200 μl/sample) was addedto a sterile 15 ml centrifuge tube and brought to 10 ml with panningbuffer. The tube was placed on the magnet for 10 min to separate thelatex. The solution was carefully removed with a 10 ml sterile pipetteas described above. The magnetic latex was resuspended in 10 ml ofpanning buffer to begin the second wash. The magnetic latex was washed atotal of 3 times with panning buffer. After the final wash, the latexwas resuspended in panning buffer to the starting volume.

Example 12 Plating M13 Phage or Cells Transformed with AntibodyPhage-display Vector Mutagenesis Reaction

[0154] The phage samples were added to 200 μL of an overnight culture ofE. coli XL1-Blue when plating on 100 mm LB agar plates or to 600 μL ofovernight cells when plating on 150 mm plates in sterile 15 ml culturetubes. After adding LB top agar (3 ml for 100 mm plates or 9 ml for 150mm plates, top agar stored at 55° C., Appendix A1, Sambrook et al.,supra.), the mixture was evenly distributed on an LB agar plate that hadbeen pre-warmed (37° C.-55° C.) to remove any excess moisture on theagar surface. The plates were cooled at room temperature until the topagar solidified. The plates were inverted and incubated at 37° C. asindicated.

Example 13 Developing Nitrocellulose Filters with Alkaline PhosphataseConjugates

[0155] After overnight incubation of nitrocellulose filters on the LBagar plates, the filters were carefully removed from the plates withmembrane forceps and incubated for 2 hr in block. After 2 hr, thefilters were incubated with goat anti-mouse kappa-AP (SouthernBiotechnology Associates, Inc, Birmingham, Ala.) for 2-4 hours. The goatanti-mouse kappa-AP was diluted into block at a final concentration of 1μg/ml. Filters were washed three times with 40 mM TRIS, 150 mM NaCl,0.05% Tween 20, pH 7.5 (TBST) (Fisher Chemical, Pittsburgh, Pa.) for 5min each. After the final wash, the filters were developed in a solutioncontaining 0.2 M 2-amino-2-methyl-1-propanol (JBL Scientific, San LuisObispo, Calif.), 0.5 M TRIS, 0.33 mg/ml nitro blue tetrazolium ((NBT)Fisher Scientific, Pittsburgh, Pa.) and 0.166 mg/ml5-bromo-4-chloro-3-indolyl-phosphate, p-toluidine salt.

Example 14 Selection of Polyclonal Antibodies to Crude SolubleCryptosporidium Antigen

[0156] The first round antibody phage was prepared as described inExample 8 using BS45 uracil template. Electroporations of mutagenesisDNA were performed yielding phage samples derived from differentimmunized mice. To create more diversity in the polyclonal library, eachphage sample was panned separately. The antibody phage (about 0.9 mL)from each electroporation was transferred to a 15 mL disposable sterilecentrifuge tube with a plug seal cap. BSA (30 μL of 300 mg/mL solution)and 1 M Tris (50 μL of 1 M stock solution, pH 8.0) were added to eachphage stock. Ten μL of 3×10⁻⁶ M biotinylated crude Cryptosporidiumsoluble antigen was added to each phage sample (Example 1A). Theantibody phage were allowed to come to equilibrium with theantigen-biotin by incubating the phage at 2-8° C. overnight.

[0157] After the incubation, the phage samples were panned with avidinmagnetic latex. The equilibrated avidin magnetic latex (see Example 11),200μL latex per sample, was incubated with the phage for 10 min at roomtemperature. After 10 min, approximately 9 mL of panning buffer wasadded to each phage sample, and the magnetic latex was separated fromthe solution using a magnet. After a ten minute separation, the unboundphage was carefully removed using a 10 mL sterile pipette. The magneticlatex was then resuspended in 10 mL of panning buffer to begin thesecond wash. The latex was washed a total of four times as describedabove. For each wash, the tubes were in contact with the magnet for 10min to separate unbound phage from the magnetic latex. After the fourthwash, the magnetic latex was resuspended in 1 mL of panning buffer andtransferred to a 1.5 mL tube.

[0158] The entire volume of magnetic latex for each sample was thenresuspended in 200 mL 2YT and was plated on 150 mm LB plates asdescribed in Example 12. The 150 mm plates were used to amplify thephage binding to the magnetic latex to generate the next round ofantibody phage. These plates were incubated at 37° C. for 4 hr, thenovernight at 20° C. After the overnight incubation, the second roundantibody phage was eluted from the 150 mm plates by pipetting 10 mL 2YTmedia onto the lawn and gently shaking the plate at room temperature for20 min. The phage samples were transferred to 15 mL disposable sterilecentrifuge tubes with a plug seal cap, and the debris from the LB platewas pelleted by centrifuging the tubes for 15 min at 3500 rpm. Thesecond round antibody phage was then transferred to a new tube.

[0159] The second round of panning was set up by diluting 50 μL of eachphage stock into 950 μL panning buffer in 15 mL disposable sterilecentrifuge tubes with plug seal cap. The biotinylated Cryptosporidiumantigen was added to each sample as described for the first round ofpanning, and the phage samples were incubated overnight at 2-8° C. Thephage samples were panned with avidin magnetic latex following theovernight incubation as described above. After washing the latexes withpanning buffer, each latex was plated on 150 mm LB agar plates. Theplates were incubated at 37° C. for 4 hr, then overnight at 20° C. Thethird round antibody phage was eluted as described above.

[0160] Panning was continued by diluting phage samples into panningbuffer as described above or by enriching the phage samples by panningusing 7 F 11 magnetic latex (described in Examples 21 and 22 of U.S.patent application Ser. No. 08/835,159, filed Apr. 4, 1997) prior tofunctional panning (see, Example 16 of U.S. patent application Ser. No.08/835,159). The progress of panning was measured by plating aliquots ofeach latex sample on 100 mm LB agar plates to determine the percentageof kappa positives. The majority of latex from each panning (99%) wasplated on 150 mm LB agar plates to amplify the phage binding to thelatex (see above). The 100 mm LB agar plates were incubated at 37° C.for 6-7 hr, after which the plates were transferred to room temperatureand nitrocellulose filters (pore size 0.45 mm, BA85 Protran, Schleicherand Schuell, Keene, N.H.) were overlayed onto the plaques. Plates withnitrocellulose filters were incubated overnight at room temperature.

[0161] After the overnight incubation, the next round antibody phage waseluted from the 150 mm plates, and the filters were developed with goatanti-mouse kappa alkaline phosphatase as described in Example 13.Individual phage samples having kappa positive percentages of greaterthan 80% on plaque lifts were pooled. The pooled phage was subclonedinto the expression vector, pBRncoH3. The subcloning was done generallyas described in Example 18 of U.S. patent application Ser. No.08/835,159.

Example 15 Expression and Purification of Recombinant Antibodies and PDIAntigen

[0162] This Example describes the expression of PDI, and recombinantantibodies that bind to PDI, using recombinant E. coli cells thatcontain genes encoding the PDI antigen of Cryptosporidium parvum orantibodies against this antigen.

[0163] A. Expression and Purification of Recombinant Antibodies

[0164] A shake flask inoculum was generated overnight from a −70° C.cell bank in an Innova 4330 incubator shaker (New Brunswick Scientific,Edison, N.J.) set at 37° C., 300 rpm. The inoculum was used to seed a 20L fermentor (Applikon, Foster City, Calif.) containing defined culturemedium (Pack et al. (1993) Bio/Technology 11: 1271-1277) supplementedwith 3 g/L L-leucine, 3 g/L L-isoleucine, 12 g/L casein digest (Difco,Detroit, Mich.), 12.5 g/L glycerol and 10 μg/ml tetracycline. Thetemperature, pH and dissolved oxygen in the fermentor were controlled at26° C., 6.0-6.8 and 25% saturation, respectively. Foam was controlled byaddition of polypropylene glycol (Dow, Midland, Mich.). Glycerol wasadded to the fermentor in a fed-batch mode. Fab expression was inducedby addition of L(+)-arabinose (Sigma, St. Louis, Mo.) to 2 g/L duringthe late logarithmic growth phase. Cell density was measured by opticaldensity at 600 nm in an UV-1201 spectrophotometer (Shimadzu, Columbia,Md.). Following run termination and adjustment of pH to 6.0, the culturewas passed twice through an M-210B-EH Microfluidizer (Microfluidics,Newton, Mass.) at 17000 psi. The high pressure homogenization of thecells released the Fab into the culture supernatant.

[0165] The first step in purification was expanded bed immobilized metalaffinity chromatography (EB-IMAC). Streamline™ chelating resin(Pharmacia, Piscataway, N.J.) was charged with 0.1 M NiCl₂ and was thenexpanded and equilibrated in 50 mM acetate, 200 mM NaCl, 10 mMimidazole, 0.01% NaN₃, pH 6.0 buffer flowing in the upward direction. Astock solution was used to bring the culture homogenate to 10 mMimidazole, following which it was diluted two-fold or higher inequilibration buffer to reduce the wet solids content to less than 5% byweight. It was then loaded onto the Streamline column flowing in theupward direction at a superficial velocity of 300 cm/hr. The cell debrispassed through unhindered, but the Fab was captured by means of the highaffinity interaction between nickel and the hexahistidine tag on the Fabheavy chain. After washing, the expanded bed was converted to a packedbed and the Fab was eluted with 20 mM borate, 150 mM NaCl, 200 mMimidazole, 0.01% NaN₃, pH 8.0 buffer flowing in the downward direction.

[0166] The second step in the purification used ion-exchangechromatography (IEC). Q Sepharose FastFlow resin (Pharmacia, Piscataway,N.J.) was equilibrated in 20 mM borate, 37.5 mM NaCl, 0.01% NaN₃, pH8.0. The Fab elution pool from the EB-IMAC step was diluted four-fold in20 mM borate, 0.01% NaN₃, pH 8.0 and loaded onto the IEC column. Afterwashing, the Fab was eluted with a 37.5-200 mM NaCl salt gradient. Theelution fractions were evaluated for purity using an Xcell II™ SDS-PAGEsystem (Novex, San Diego, Calif.) prior to pooling. Finally, the Fabpool was concentrated and diafiltered into 20 mM borate, 150 mM NaCl,0.01% NaN₃, pH 8.0 buffer for storage. This was achieved in a SartoconSliceTm system fitted with a 10,000 MWCO cassette (Sartorius, Bohemia,N.Y.). The final purification yields were typically 50%. Theconcentration of the purified Fab was measured by UV absorbance at 280nm, assuming an absorbance of 1.6 for a 1 mg/ml solution.

[0167] B. Expression and Purification of PDI

[0168] A shake flask inoculum was generated overnight from a −70° C.cell bank in an incubator shaker set at 37° C., 300 rpm. The cells werecultured in a defined medium described above. The inoculum was used toseed a 2 L Tunair shake flask (Shelton Scientific, Shelton, Conn.) whichwas grown at 37° C., 300 rpm. Expression was induced by addition ofL(+)-arabinose to 2 g/L during the logarithmic growth phase, followingwhich, the flask was maintained at 23° C., 300 rpm. Following batchtermination, the culture was passed through an M-10 Y Microfluidizer(Microfluidics, Newton, Mass.) at 17000 psi. The homogenate wasclarified in a J2-21 centrifuge (Beckman, Fullerton, Calif.).

[0169] Purification employed immobilized metal affinity chromatography.Chelating Sepharose FastFlow™ resin (Pharmacia, Piscataway, N.J.) wascharged with 0.1 M NiCl₂ and equilibrated in 20 mM borate, 150 mM NaCl,10 mM imidazole, 0.01% NaN₃, pH 8.0 buffer. A stock solution was used tobring the culture supernatant to 10 mM imidazole and 2-mercaptoethanolwas added to 1 mM. The culture supernatant was then mixed with the resinand incubated in the incubator shaker set at room temperature, 150-200rpm. The antigen was captured by means of the high affinity interactionbetween nickel and the hexahistidine tag on the antigen. The culturesupernatant and resin mixture is poured into a chromatography column.After washing, the antigen was eluted with 20 mM borate, 150 mM NaCl,200 mM imidazole, 1 mM 2-mercaptoethanol, 0.01% NaN₃, pH 8.0 buffer. Theantigen pool was concentrated in a stirred cell fitted with a 10,000MWCO membrane (Amicon, Beverly, Mass.). It was then dialyzed overnightinto 20 mM borate, 150 mM NaCl, 0.01% NaN₃, pH 8.0 for storage, using12-14,000 MWCO dialysis tubing. The purified antigen was evaluated forpurity by SDS-PAGE analysis. The concentration of the PDI antigen ismeasured by UV absorbance at 280 nm, assuming an absorbance of 0.66 fora one mg/ml solution.

Example 16 Selection of Monoclonal Antibodies to Crude CryptosporidiumAntigen from the Polyclonal Antibody

[0170] It was desired to have a monoclonal/polyclonal antibody pair toone specific antigen. Individual colonies were picked off the LB agartetracycline plates from the subcloned phage plated in Example 14 into2YT media and tetracycline (10 μg/mL). The cultures were grown overnightat 37° C., 300 rpm. These monoclonal antibodies were expressed andpurified as described in Example 15, and biotinylated as described inExample 10. The polyclonal antibody (Example 14) was conjugated toalkaline phosphatase as described in Example 19.

[0171] The sensitivity of the monoclonal antibodies was determined byperforming a sandwich assay using the biotinylated monoclonal antibodiesand the alkaline phosphatase-conjugated polyclonal antibodies. Assayswere performed with streptavidin coated plates such as Reacti-Bind™streptavidin coated polystyrene 96 well plates (Pierce Chemical,Rockford, Ill.). After washing the 96 well plate with a plate washersuch as the Skan Washer (Skatron Instruments, Sterling, Va.),biotinylated monoclonal antibody (50μL of 2.5μg/mL stock diluted inblock) was added to 8 wells. The plate was incubated at room temperaturefor 1 hr. The plate was then washed, after which Cryptosporidium solubleantigen (50 μL) was added in duplicate to the biotinylated monoclonalwells at different concentrations. The approximate concentrations ofcrude antigen were 5 μg/mL, 0.5 μg/mL, 0.05 μg/mL, and 0. Antigen wasincubated for 1 hr at room temperature, after which the plate waswashed. The polyclonal antibody-alkaline phosphatase conjugate (50 μL of2.5 μg/mL diluted in block) was added and incubated at room temperaturefor 1 hr.

[0172] After 1 hr, the plate was washed and developed using the ELISAAmplification System (Gibco BRL, Gaithersburg, Md.) according to themanufacturer's instructions. Monoclonal antibody CP.2 had the highestsignal, which was slightly above background at 0.05 μg/mL. Thismonoclonal was chosen to make a complementary polyclonal, and themonoclonal was used to screen a Cryptosporidium cDNA library to identifythe antigen.

Example 17 Selection and Cloning of Polyclonal Antibody Complementary toCP.2 Monoclonal Antibody

[0173] Phage samples enriched for binding to crude Cryptosporidiumantigen as described in Example 14 were pooled using an equal number ofphage from each sample. Biotinylated CP.2 monoclonal antibody (12 μl,10-6M) and soluble crude Cryptosporidium antigen (12 μl, about 2 mg/mL)were mixed and incubated for 10 min at room temperature. Twenty μl ofCP.2 biotin/antigen was added to the phage sample, and the sample wasincubated overnight at 2-8° C. The sample was panned with avidinmagnetic latex and plated as described in Example 14. The eluted phagewas panned a second time as described using biotinylated CP.2/crudeCryptosporidium antigen. The phage eluted after the second round ofpanning were subcloned as described in Example 18 of U.S. patentapplication Ser. No. 08/835,159. This polyclonal was designatedSCPc.4.PC.

Example 18 Microtiter Plate Assay Sensitivity

[0174] The sensitivity of the monoclonal/polyclonal antibody pair wasdetermined with a sandwich assay using biotinylated CP.2 and alkalinephosphatase conjugated SCPc.4.PC. After washing the 96 well plate with aplate washer (see Example 16), biotinylated CP.2 (50 μL of 2.5 μg/mLdiluted in block) was added to 12 wells. The plate was incubated at roomtemperature for 1 hr. The plate was washed, then purified PDI (50 μL)was added in duplicate to the biotinylated monoclonal wells at fivedifferent concentrations of antigen (see Table 3) and block was added tothe last two wells for the blank. Antigen was incubated for 1 hr at roomtemperature, after which the plate was washed. The complementarypolyclonal alkaline phosphatase conjugate (SCPc.4.PC, 50 μL of 2.5 μg/mLdiluted in block) was added and incubated at room temperature for lhr.After 1 hr, the plate was washed and developed using the ELISAAmplification System according to the manufacturer's instructions. Thesignal was read at 490 nm using a microplate reader (Molecular Devices,Sunnyvale, Calif.). Table 3 lists the signal at 490 nm versus theconcentration of PDI antigen. TABLE 3 concentration of PDI antigenversus signal at 490 nm (endpoint reading) for the antibody pairCP.2/SCPc.4.PC Concentration (ng/mL) Absorbance (490 nm) 0 0.055 3.10.71 6.25 1.203 12.5 2.07 25 2.687 50 2.996

Example 19 Preparation and Testing of Device for Detecting C. parvumInfection

[0175] This Example describes the preparation and testing of a devicefor detecting Cryptosporidium parvum infection. The device employs therecombinant polyclonal antibody to immobilize C. parvum PDI on a solidsupport, and a recombinant monoclonal antibody to detect the presence ofimmobilized PDI.

[0176] A. Preparation of Antibody-alkaline Phosphatase Conjugates foruse as Detection Reagents.

[0177] Detection reagents for use in the assay were prepared byconjugating alkaline phosphatase to antibodies for protein disulfideisomerase. The recombinant monoclonal antibody CP.2 was used to detectprotein disulfide isomerase. Alkaline phosphatase (AP, CalzymeLaboratories, San Luis Obispo, Calif.) was dialyzed against a minimum of100 volumes of column buffer (50 mM potassium phosphate, 10 mM borate,150 mM NaCl, IMM MgSO₄, pH 7.0) at 2-8° C. for a minimum of four hoursand the buffer was changed at least twice prior to use of the AP. Afterthe AP was removed from dialysis and brought to room temperature, theconcentration was determined by determining the A₂₈₀, with an absorbanceof 0.77 indicating a 1 mg/ml solution. The AP was diluted to 5 mg/mlwith column buffer.

[0178] For crosslinking the AP to the antibody, AP was first linked tosuccinimidyl 4-(N-maleimidomethyl cyclohexane-1-carboxylate (SMCC,Pierce Chemical Co., Rockford Ill.) using a 20:1 ratio of SMCC:AP. SMCCwas dissolved in acetonitrile at 20 mg/ml and diluted by a factor of 84when added to AP while vortexing or rapidly stirring. The solution wasallowed to stand at room temperature for 90 minutes before the unreactedSMCC and low molecular weight reaction products were separated from theAP using gel filtration chromatography (G-50 Fine, Pharmacia Biotech,Piscataway, N.J.) in a column equilibrated with column buffer.

[0179] Recombinant antibodies were reacted with 1 mM dithiothreitol(DTT, Calbiochem, San Diego, Calif.) for 30 minutes at room temperatureto reduce a cysteine residue present near the carboxy terminus of theheavy chain constant region. The DTT was separated from the antibody bygel filtration chromatography using G50 Fine in column buffer withoutMgSO₄ but containing 0.1 mM ethylenediaminetetraacetic acid (EDTA,Fisher Scientific, Pittsburgh, Pa.). The AP and the antibody were mixedtogether in a molar ratio of 6 antibodies to one alkaline phosphataseand the conjugation reaction was allowed to continue for one hour atroom temperature. To stop the conjugation, 2-mercaptoethanol was addedto 1 mM final concentration to the conjugate solution and reacted for 5minutes followed by the addition of N-ethyl maleimide to 2 mM finalconcentration. The conjugate was purified by gel filtrationchromatography using SEPHACRYL™ S-200 HR (Pharmacia Biotech, Piscataway,N.J.). The free antibody was excluded from the conjugate pool which wasdiluted for use in immunoassays in a conjugate diluent containing 1%bovine serum albumin (from 30% BSA, Bayer, Kankakee. Ill.), 2% casein(Hammersten grade, Research Organics, Cleveland, Ohio), 100 mM trehalose(Aldrich Chemical Co., Milwaukee, Wis.), 50 mM potassium phosphate, 150mM sodium chloride, 1 mM MgSO₄, 0.1 mM ZnCl₂, 0.1% polyvinyl alcohol(80% hydrolyzed, Aldrich Chemical Co., Milwaukee Wis.), pH 7.0.

[0180] B. Preparation of Antibody-casein Conjugates for use as CaptureReagents

[0181] Capture reagents for protein disulfide isomerase were prepared asfollows. Where recombinant antibodies were used as anchor moieties, theantibodies were first conjugated to casein. Casein was dissolved indeionized water at 2.5% solids by stirring it at 37-45° C. while addingconcentrated potassium hydroxide to keep the pH of the solution between7 and 8. After the pH had stabilized at 7.0, the casein was diluted withdeionized water to a final A₂₈₀ of 10. The casein solution was subjectedto tangential flow filtration through an ultrafiltration membrane with amolecular weight cut-off of 300,000 in order to exclude aggregatedprotein from the filtrate. The casein filtrate was concentrated to afinal A₂₈₀ of approximately 10 by ultrafiltration. A solution of SMCCwas prepared at 20 mg/ml (60 mM) in acetonitrile; this was diluted intothe casein solution to a final concentration of 2 mM SMCC. The solutionwas allowed to stand for 90 minutes at room temperature and then wassubjected to gel filtration chromatography in a column containing G50Fine equilibrated in column buffer in order to separate the protein fromthe reactants. The casein was mixed with recombinant antibody SCPc.4.PCthat had been reacted with 1 mM DTT and subjected to gel filtrationchromatography to remove the DTT as described in Example 19A above. Theantibody was mixed with the casein in a 4:1 molar ratio and the reactionwas allowed to proceed for one hour at room temperature before theconjugation was stopped as described above. The conjugate solution wassubjected to gel filtration chromatography in a column containingSEPHACRYL™ S-200 HR in order to separate the conjugated antibody fromthe unconjugated antibody. The conjugated antibody was concentratedusing an ultrafiltration membrane and subjected to dialysis vs.borate-buffered saline (BBS, 20 mM borate, 150 mM sodium chloride, 0.02%sodium azide, pH 8.2) and stored in BBS until immobilization on nylonmembranes.

[0182] C. Preparation of Assay Devices

[0183] The assays were performed using capture reagents that wereimmobilized on nylon membranes. Recombinant Fab antibodies wereconjugated to casein as described above prior to immobilization. Theantibodies were immobilized on the nylon membranes (5 μm pore size;IMMUNODYNE™, Pall Corporation, Glen Cove, N.Y.) in a continuous processby pumping an antibody solution directly onto the membrane while themembrane was moved past a stationary nozzle which dispensed the antibodysolution at a flow rate controlled by the pump. The antibody solutiontypically contained antibody at a concentration between 1 and 5 mg/ml ina buffer containing 20 mM borate, 150 mM sodium chloride, 0.02% sodiumazide, and 10% trehalose, pH 8.2.

[0184] Each antibody was immobilized in a line approximately 0.040inches wide, such that approximately 36 μL of antibody solution wasrequired per linear foot of membrane. The antibody solution applied tothe membrane was dried prior to blocking the entire membrane bysaturating it with a solution containing 2% casein, 40% STABILICOAT™(Bio-metric Systems, Eden Prairie, Minn.), 0.25% TRITON X-100™ (SigmaChemical Co., St. Louis, Mo.) and drying the membrane in a drying tunnelor in a dry room. The antibody can also be applied in spots by applyinga volume of approximately 1 μL of antibody solution to the membrane atthe desired location prior to blocking and drying the membrane.Generally, several lines of immobilized antibody were placed on amembrane in this manner and the membrane was cut perpendicular to thedirection of the antibody lines for placement in the assay devices.

[0185] The cut membrane pieces were ultrasonically welded to an openingin a plastic device top (see FIG. 1A—top view, FIG. 1B—side section, andFIG. 1C—end view) which was then ultrasonically welded to a plasticbottom piece (see FIG. 2A—top view, FIG. 2B—side section, and FIG.2C—end view) having grooves cut into its upper surface. The contactbetween the membrane and the two plastic pieces resulted in a network ofcapillary channels that caused fluids added to the membrane to flowthrough the membrane and into the capillary network between the twoplastic pieces. Such devices are described in European PatentApplication No. 447154.

[0186] For the immunoassay of protein disulfide isomerase, a total ofthree lines of antibody were immobilized on the membrane. The top linein the device was a positive control for the immunoassay of proteindisulfide isomerase. The antibody solution used in the immobilizationstep for the positive control contained protein disulfide isomerase atapproximately 1 μg/ml mixed with the SCPc.4.PC-casein conjugate atapproximately 1 mg/ml. The next line on the membrane was for the captureand detection of protein disulfide isomerase. The solution used toimmobilize the antibody for protein disulfide isomerase containedapproximately 2 mg/ml of the SCPc.4.PC antibody conjugated to casein.The last line of immobilized antibody on the device was a negativecontrol line; the antibody solution used to apply this line to themembrane contained a recombinant polyclonal antibody (2 mg/ml) that wasspecific for an antigen not found in C. parvum.

[0187] For filtering samples prior to performing the assays, disposablefilter devices were constructed using standard 10-cc plastic syringes.Disks of filter material were cut to a diameter that would allow thedisk to be placed into the barrel of the syringe so that sufficientcontact was created between the syringe barrel and the edge of thefilter disk. This prevented fluids from bypassing the filter materialwhen liquid samples were forced through the filter by the plunger. Atthe bottom of the syringe closest to the outlet was a disk of glassfiber filter (GF/F, 0.7 μm, Whatman, Clifton, N.J.) followed by a diskof porous plastic (Porex Technologies, Fairburn, Ga.). The next twodisks of filter material were both cut from CELLUPORE™ filter grade 850material (Cellulo Co., Fresno, Calif.). The next disk of filter materialwas cut from CELLUPORE™ filter grade 315 material (Cellulo Co., Fresno,Calif.). The uppermost filter element in the syringe barrel was a bondedcellulose acetate material (American Filtrona, Richmond, Va.) thatserved as a prefilter for the filter elements described previously. Analternative filter device that contains essentially the same elements isthe AUTOVIAL™ (Whatman, Clifton, N.J.) which is a disposable syringethat has a GMF glass fiber filter with a rating of 0.45 μm alreadyconnected to the end of the syringe. The other filter elements describedabove are placed in the barrel of the AUTOVIAL™ in the same order.

[0188] D. Immunoassay of Protein Disulfide Isomerase

[0189] Stool samples (approximately 0.5 g or 0.5 ml) were dilutedtenfold with sample diluent containing 1% casein, 100 mM potassiumphosphate, 150 mM sodium choride, 0.1% Dow 193 surfactant (Dow Coming,Midland, Mich.), 0.1% bovine IgG (Sigma Chemical Co., St. Louis, Mo.),0.1% sodium azide, pH 7.0, and then poured into the barrel of a filterdevice. The syringe plunger was inserted into the filter device andpressed down to expel the filtered sample through the end of the syringeinto a tube. Using a disposable transfer pipet, 0.5 ml of sample wastaken from the tube and transferred to the exposed membrane in theimmunoassay device described above.

[0190] After the sample drained through the membrane in the device, theantibody CP.2 conjugated to alkaline phosphatase was applied in a volumeof 140 μL and incubated for 3 minutes. The antibody conjugateconcentration was approximately 10 μg/ml. After the incubation, sixdrops of wash solution containing 100 mM Tris (hydroxymethyl)aminomethane (TRIS, Fisher Scientific, Pittsburgh, Pa.), 150 mM sodiumchloride, 0.5% Dow 193 surfactant, 0.1% sodium azide, and 20 mg/l ofnitro blue tetrazolium (NBT) were applied from a dropper bottle. Afterthe wash drained into the membrane, another six drops of wash solutionwere applied and allowed to drain. Three drops of substrate solutioncontaining 10 mM indoxyl phosphate (JBL Scientific, San Luis Obispo,Calif.), 200 mM 2-amino-2-methyl-1-propanol (JBL Scientific, San LuisObispo, Calif.), 500 mM TRIS, pH 10.2, were added from a dropper bottleand the device was incubated for five minutes at room temperature.

[0191] At the end of the incubation time, the presence of any visuallydetectable purple to black lines was noted. The positive control zonedescribed above developed a clearly visible line that resulted from thebinding of the antibody-alkaline phosphatase conjugate to theimmobilized complex of antigen and antibody. Control samples containingprotein disulfide isomerase spiked from purified preparations ofrecombinant protein to concentrations of 2 ng/ml or greater resulted ina visible line at the zone for the detection of this antigen. Thenegative control zone for the detection of non-specific binding ofreagents developed a visible response for less than 1% of the clinicalsamples tested. When tested again using ¼ of the initial sample volume,no visible response was observed at the negative control zone for any ofthe samples.

[0192] E. Sensitivity of Assay with Purified Antigen

[0193] The purified recombinant antigen was serially diluted in asolution containing 1% bovine serum albumin, 10 mM3-(N-morpholino)propanesulfonic acid (Fisher Scientific, Pittsburgh,Pa.), 150 mM sodium chloride, and 0.1% sodium azide, pH 7.0, anddilutions were tested in replicates of ten using the same procedureemployed with stool samples, a tenfold dilution of a 0.5-ml samplefollowed by filtration of the diluted sample. The lowest concentrationof the antigen that consistently produced a positive visual response atthe detection zone on the membrane was determined to be the limit ofsensitivity of the assay. For protein disulfide isomerase, this wasfound to be 3 ng/ml.

[0194] F. Clinical Sensitivity and Specificity of the Assay

[0195] The clinical sensitivity and specificity of the assay wasdetermined by testing 444 samples obtained from a patient population inMexico and Peru. The results were compared to those obtained with astandard ova and parasite examination and with a commercially availableenzyme-labeled microtiter plate immunoassay (Alexon ProSpecTCryptosporidium Microplate Assay). Discrepancies between methods wereresolved by comparing the three results for the discrepant sample. Sinceno method exists that can unequivocally identify the presence of theorganism in samples, when two of the three methods produced the sameresult, that result was judged to be the correct result for that sample.Clinical sensitivity, specificity, positive predictive value andnegative predictive value were calculated as described in the TietzTextbook of Clinical Chemistry (second edition, page 496). The resultsare shown in Table 4-Table 6. The assay for protein disulfide isomerasewas shown to be more sensitive than traditional ova and parasite methodsfor the detection of C. parvum in clinical samples. Furthermore, theassay of the present invention was substantially equivalent to acommercially available immunoassay that detects an unspecified antigenor mixture of antigens. TABLE 4 O & P Examination + − Total Triage ® C.parvum + 53  7  60 −  5 379 384 Total 58 386 444 Sensitivity 91.4%Specificity 98.2% Positive Predictive 88.3% Value Negative Predictive98.7% Value

[0196] TABLE 5 Alexon + − Total Triage ® C. parvum + 58  2  60 −  7 377384 Total 65 379 444 Sensitivity 89.2% Specificity 99.5% PositivePredictive 96.7% Value Negative Predictive 98.2% Value

[0197] TABLE 6 Resolved + − Total Triage ® C. parvum + 59  1  60 −  5379 384 Total 64 380 444 Sensitivity 92.2% Specificity 99.7% PositivePredictive 98.3% Value Negative Predictive 98.7% Value

[0198] It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims. All publications, patents,and patent applications cited herein are hereby incorporated byreference for all purposes.

What is claimed is:
 1. A method of diagnosing infection of a mammal by aCryptosporidium species, the method comprising: contacting a stoolsample obtained from the mammal with a capture reagent which binds toCryptosporidium protein disulfide isomerase, wherein the capture reagentforms a complex with the protein disulfide isomerase if the proteindisulfide isomerase is present in the stool sample; and detectingwhether protein disulfide isomerase is bound to the capture reagent,wherein the presence of protein disulfide isomerase is indicative ofCryptosporidium infection of the mammal.
 2. The method of claim 1,wherein the protein disulfide isomerase comprises an amino acid sequenceat least ten consecutive amino acids of which are substantiallyidentical to a subsequence of an amino acid sequence AWFCGTNEDFAKYASNIRKVAADYR EKYAFVF (SEQ ID NO: 3).
 3. The method of claim 2, whereinthe protein disulfide isomerase has an amino acid sequence that issubstantially identical to the amino acid sequence of SEQ ID NO:2. 4.The method of claim 1, wherein the capture reagent comprises an antibodywhich binds to protein disulfide isomerase.
 5. The method of claim 4,wherein the antibody is a recombinant antibody.
 6. The method of claim5, wherein the antibody is a recombinant polyclonal antibody.
 7. Themethod of claim 6, wherein the recombinant polyclonal antibody isSCPc.4.PC.
 8. The method of claim 1, wherein the capture reagent isimmobilized on a solid support.
 9. The method of claim 8, wherein thecapture reagent is immobilized on the solid support prior to contactingthe capture reagent with the test sample.
 10. The method of claim 1,wherein the detection of the protein disulfide isomerase is performed bycontacting the protein disulfide isomerase with a detection reagentwhich binds to the protein disulfide isomerase.
 11. The method of claim10, wherein the detection reagent comprises an antibody which binds toprotein disulfide isomerase.
 12. The method of claim 10, wherein thedetection reagent comprises a detectable label.
 13. The method of claim12, wherein the detectable label is selected from the group consistingof a radioactive label, a fluorophore, a dye, an enzyme, and achemiluminescent label.
 14. A kit for diagnosing infection of a mammalby an Cryptosporidium species, the kit comprising: a solid support uponwhich is immobilized a capture reagent which binds to a proteindisulfide isomerase of Cryptosporidium parvum; and a detection reagentwhich binds to the protein disulfide isomerase.
 15. The kit according toclaim 14, wherein the kit further comprises a positive control thatcomprises a protein disulfide isomerase.
 16. The kit according to claim15, wherein the protein disulfide isomerase comprises an amino acidsequence of which at least ten consecutive amino acids are substantiallyidentical to an amino acid sequence AWFCGTNEDFAKYASNIRKVAADYR EKYAFVF(SEQ ID NO: 3).
 17. A monoclonal antibody that specifically binds to aprotein disulfide isomerase of Cryptosporidium parvum, wherein themonoclonal antibody is CP.2.
 18. A recombinant polyclonal antibodypreparation that specifically binds to protein disulfide isomerase ofCryptosporidium parvum.
 19. The recombinant polyclonal antibodypreparation of claim 18, wherein the protein disulfide isomerasecomprises an amino acid sequence of which at least ten consecutive aminoacids are substantially identical to an amino acid sequenceAWFCGTNEDFAKYASNIRKVAADYREKYAFVF (SEQ ID NO: 3).
 20. The recombinantpolyclonal antibody preparation of claim 18, wherein the antibodypreparation is SCPc.4.PC.
 21. An isolated protein disulfide isomerasepolypeptide which comprises an amino acid sequence of which at least tenconsecutive amino acids are substantially identical to a subsequence ofan amino acid sequence AWFCGTNEDFAKYASNIRKVAADYR EKYAFVF (SEQ ID NO: 3).22. The protein disulfide isomerase polypeptide of claim 21, wherein thepolypeptide comprises an amino acid sequence that is substantiallyidentical to the amino acid sequence of SEQ ID NO:
 2. 23. The proteindisulfide isomerase polypeptide of claim 21, wherein the polypeptidecomprises an amino acid sequence that is substantially identical to anamino acid sequence AWFCGTNEDFAKYASNIRKVAADYREKYAFVF (SEQ ID NO: 3). 24.The protein disulfide isomerase polypeptide of claim 23, wherein thepolypeptide comprises an amino acid sequence of SEQ ID NO:
 3. 25. Theprotein disulfide isomerase polypeptide of claim 24, wherein thepolypeptide comprises the amino acid sequence of SEQ ID NO:
 2. 26. Anisolated nucleic acid that comprises a polynucleotide sequence thatencodes a polypeptide that comprises an amino acid sequence of which atleast ten consecutive amino acids are substantially identical to asubsequence of an amino acid sequence AWFCGTNEDFAKYASNIRKVAADYREKYAFVF(SEQ ID NO: 3).
 27. The nucleic acid of claim 26, wherein the nucleicacid comprises a translation initiation codon that is in frame withcodons that encode the amino acid sequence set forth in SEQ ID NO: 3.28. The nucleic acid of claim 27, wherein the nucleic acid comprises apolynucleotide sequence of SEQ ID NO:
 1. 29. The nucleic acid of claim26, wherein the nucleic acid is operably linked to a promoter.
 30. Thenucleic acid of claim 29, wherein the nucleic acid comprises anexpression cassette.
 31. A recombinant cell that comprises an expressioncassette of claim 30.